T cell-independent antibody-mediated clearance of polyoma virus in T cell-deficient mice

Polyomavirus (PyV) infection of SCID mice, which lack functional T and B cells, leads to a lethal acute myeloproliferative disease (AMD) and to high levels of virus replication in several organs by two wk after infection. This is in contrast to infection of T cell-deficient athymic nude mice, which are resistant to acute PyV-induced disease and poorly replicate the virus in their organs. This major difference in the virus load and in the outcome of PyV infection between SCID and nude mice suggested that an efficient, T cell-independent antiviral mechanism operates in T cell-deficient, PyV infected mice. To investigate this possibility, mice with different genetically engineered T and/or B cell deficiencies and SCID mice adoptively reconstituted with B and/or T cells were infected with PyV. The results indicated that the presence of B cells in the absence of T cells protected mice from the AMD, and this was accompanied by a major reduction of PyV in all organs tested. Sera from PyV-infected T cell receptor (TCR) alpha beta knockout or TCR alpha beta gamma delta knockout mice contained IgG2a antibodies to PyV. Sera or purified immunoglobulin fractions from PyV-infected TCR alpha beta knockout mice protected SCID mice from the PyV-induced AMD. To our knowledge, this is the first report of an effective T cell-independent antibody response clearing a virus and changing the outcome of infection from 100% mortality to 100% survival

Scaffolded Antigens in Yeast Cell Particle Vaccines Provide Protection against Systemic Polyoma Virus Infection

Background. U65, a self-aggregating peptide scaffold, traps fused protein antigens in yeast cells. Conversion to Yeast Cell Particle (YCP) vaccines by partial removal of surface mannoproteins exposes beta-glucan, mediating efficient uptake by antigen-presenting cells (APCs). YCP vaccines are inexpensive, capable of rapid large-scale production and have potential for both parenteral and oral use. Results. YCP processing by alkaline hydrolysis exposes up to 20% of the glucan but converts scaffolded antigen and internal yeast proteins into a common aggregate, preventing selective yeast protein removal. For U65-green fluorescent protein (GFP) or U65-Apolipoprotein A1 (ApoA1) subcutaneous vaccines, maximal IgG responses in mice required 10% glucan exposure. IgG responses to yeast proteins were 5-fold lower. Proteolytic mannoprotein removal produced YCPs with only 6% glucan exposure, insufficiently porous for selective removal of even native yeast proteins. Vaccine efficacy was reduced 10-fold. Current YCP formulations, therefore, are not suitable for human use but have considerable potential for use in feed animal vaccines. Significantly, a YCP vaccine expressing a GFP fusion to VP1, the murine polyoma virus major capsid protein, after either oral or subcutaneous administration, protected mice against an intraperitoneal polyoma virus challenge, reducing viral DNA levels in spleen and liver by \u3e 98%

NK cells and gammadelta T cells mediate resistance to polyomavirus-induced tumors

NK and gammadelta T cells can eliminate tumor cells in many experimental models, but their effect on the development of tumors caused by virus infections in vivo is not known. Polyomavirus (PyV) induces tumors in neonatally infected mice of susceptible strains and in adult mice with certain immune deficiencies, and CD8+ alphabeta T cells are regarded as the main effectors in anti-tumor immunity. Here we report that adult TCRbeta knockout (KO) mice that lack alphabeta but have gammadelta T cells remain tumor-free after PyV infection, whereas TCRbeta x delta KO mice that lack all T cells develop tumors. In addition, E26 mice, which lack NK and T cells, develop the tumors earlier than TCRbeta x delta KO mice. These observations implicate gammadelta T and NK cells in the resistance to PyV-induced tumors. Cell lines established from PyV-induced tumors activate NK and gammadelta T cells both in culture and in vivo and express Rae-1, an NKG2D ligand. Moreover, these PyV tumor cells are killed by NK cells in vitro, and this cytotoxicity is prevented by treatment with NKG2D-blocking antibodies. Our findings demonstrate a protective role for NK and gammadelta T cells against naturally occurring virus-induced tumors and suggest the involvement of NKG2D-mediated mechanisms

Long-lasting T cell-independent IgG responses require MyD88-mediated pathways and are maintained by high levels of virus persistence

Many viruses induce acute T cell-independent (TI) B cell responses due to their repetitive epitopes and the induction of innate cytokines. Nevertheless, T cell help is thought necessary for the development of long-lasting antiviral antibody responses in the form of long-lived plasma cells and memory B cells. We found that T cell-deficient (T cell receptor beta and delta chain [TCRbetadelta] knockout [KO]) mice persistently infected with polyomavirus (PyV) had long-lasting antiviral serum IgG, and we questioned whether they could generate TI B cell memory. TCRbetadelta KO mice did not form germinal centers after PyV infection, lacked long-lived IgG-secreting plasma cells in bone marrow, and did not have detectable memory B cell responses. Mice deficient in CD4(+) T cells had a lower persisting virus load than TCRbetadelta KO mice, and these mice had short-lived antiviral IgG responses, suggesting that a high virus load is required to activate naive B cells continuously, and maintain the long-lasting serum IgG levels. Developing B cells in bone marrow encounter high levels of viral antigens, which can cross-link both their B cell receptor (BCR) and Toll-like receptors (TLRs), and this dual engagement may lead to a loss of their tolerance. Consistent with this hypothesis, antiviral serum IgG levels were greatly diminished in TCRbetadelta KO/MyD88(-/-) mice. We conclude that high persisting antigen levels and innate signaling can lead to the maintenance of long-lasting IgG responses even in the absence of T cell help. IMPORTANCE: Lifelong control of persistent virus infections is essential for host survival. Several members of the polyomavirus family are prevalent in humans, persisting at low levels in most people without clinical manifestations, but causing rare morbidity/mortality in the severely immune compromised. Studying the multiple mechanisms that control viral persistence in a mouse model, we previously found that murine polyomavirus (PyV) induces protective T cell-independent (TI) antiviral IgG. TI antibody (Ab) responses are usually short-lived, but T cell-deficient PyV-infected mice can live for many months. This study investigates how protective IgG is maintained under these circumstances and shows that these mice lack both forms of B cell memory, but they still have sustained antiviral IgG responses if they have high levels of persisting virus and intact MyD88-mediated pathways. These requirements may ensure life-saving protection against pathogens even in the absence of T cells, but they prevent the continuous generation of TI IgG against harmless antigens

Kinematic biomechanical assessment of human articular cartilage transplants in the knee using 3-T MRI: an in vivo reproducibility study

The aims of this study were to examine the clinical feasibility and reproducibility of kinematic MR imaging with respect to changes in T (2) in the femoral condyle articular cartilage. We used a flexible knee coil, which allows acquisition of data in different positions from 40 degrees flexion to full extension during MR examinations. The reproducibility of T (2) measurements was evaluated for inter-rater and inter-individual variability and determined as a coefficient of variation (CV) for each volunteer and rater. Three different volunteers were measured twice and regions of interest (ROIs) were selected by three raters at different time points. To prove the clinical feasibility of this method, 20 subjects (10 patients and 10 age- and sex-matched volunteers) were enrolled in the study. Inter-rater variability ranged from 2 to 9 and from 2 to 10% in the deep and superficial zones, respectively. Mean inter-individual variability was 7% for both zones. Different T (2) values were observed in the superficial cartilage zone of patients compared with volunteers. Since repair tissue showed a different behavior in the contact zone compared with healthy cartilage, a possible marker for improved evaluation of repair tissue quality after matrix-associated autologous chondrocyte transplantation (MACT) may be available and may allow biomechanical assessment of cartilage transplants

The MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) 2.0 Knee Score and Atlas

Objective Since the first introduction of the MOCART (Magnetic Resonance Observation of Cartilage Repair Tissue) score, significant progress has been made with regard to surgical treatment options for cartilage defects, as well as magnetic resonance imaging (MRI) of such defects. Thus, the aim of this study was to introduce the MOCART 2.0 knee score — an incremental update on the original MOCART score — that incorporates this progression. Materials and Methods The volume of cartilage defect filling is now assessed in 25% increments, with hypertrophic filling of up to 150% receiving the same scoring as complete repair. Integration now assesses only the integration to neighboring native cartilage, and the severity of surface irregularities is assessed in reference to cartilage repair length rather than depth. The signal intensity of the repair tissue differentiates normal signal, minor abnormal, or severely abnormal signal alterations. The assessment of the variables "subchondral lamina," "adhesions," and "synovitis" was removed and the points were reallocated to the new variable "bony defect or bony overgrowth." The variable "subchondral bone" was renamed to "subchondral changes" and assesses minor and severe edema-like marrow signal, as well as subchondral cysts or osteonecrosis-like signal. Overall, a MOCART 2.0 knee score ranging from 0 to 100 points may be reached. Four independent readers (two expert readers and two radiology residents with limited experience) assessed the 3 T MRI examinations of 24 patients, who had undergone cartilage repair of a femoral cartilage defect using the new MOCART 2.0 knee score. One of the expert readers and both inexperienced readers performed two readings, separated by a four-week interval. For the inexperienced readers, the first reading was based on the evaluation sheet only. For the second reading, a newly introduced atlas was used as an additional reference. Intrarater and interrater reliability was assessed using intraclass correlation coefficients (ICCs) and weighted kappa statistics. ICCs were interpreted according to Koo and Li; weighted kappa statistics were interpreted according to the criteria of Landis and Koch. Results The overall intrarater (ICC = 0.88, P < 0.001) as well as the interrater (ICC = 0.84, P < 0.001) reliability of the expert readers was almost perfect. Based on the evaluation sheet of the MOCART 2.0 knee score, the overall interrater reliability of the inexperienced readers was poor (ICC = 0.34, P < 0.019) and improved to moderate (ICC = 0.59, P = 0.001) with the use of the atlas. Conclusions The MOCART 2.0 knee score was updated to account for changes in the past decade and demonstrates almost perfect interrater and intrarater reliability in expert readers. In inexperienced readers, use of the atlas may improve interrater reliability and, thus, increase the comparability of results across studies

Selection of restriction endonucleases using artificial cells

We describe in this article an in vitro system for the selection of restriction endonucleases using artificial cells. The artificial cells are generated in the form of a water-in-oil emulsion by in vitro compartmentalization. Each aqueous compartment contains a reconstituted transcription/translation mix along with the dispersed DNA templates. In the compartments containing endonuclease genes, an endonuclease expressed in vitro cleaves its own DNA template adjacent to the gene, leaving a sticky end. The pooled DNA templates are then ligated to an adaptor with a compatible end. The endonuclease genes are then enriched by adaptor-specific PCR on the ligation mix. We demonstrate that the system can achieve at least 100-fold enrichment in a single round of selection. It is sensitive enough to enrich an active endonuclease gene from a 1:105 model library in 2–3 rounds of selection. Finally, we describe experiments where we selected endonuclease genes directly from a bacterial genomic DNA source in three rounds of selections: the known PstI gene from Providencia stuartii and the new TspMI gene from Thermus sp. manalii. This method provides a unique tool for cloning restriction endonuclease genes and has many other potential applications

A distinct role for B1b lymphocytes in T cell-independent immunity

Pathogenesis of infectious disease is not only determined by the virulence of the microbe but also by the immune status of the host. Vaccination is the most effective means to control infectious diseases. A hallmark of the adaptive immune system is the generation of B cell memory, which provides a long-lasting protective antibody response that is central to the concept of vaccination. Recent studies revealed a distinct function for B1b lymphocytes, a minor subset of mature B cells that closely resembles that of memory B cells in a number of aspects. In contrast to the development of conventional B cell memory, which requires the formation of germinal centers and T cells, the development of B1b cell-mediated long-lasting antibody responses occurs independent of T cell help. T cell-independent (TI) antigens are important virulence factors expressed by a number of bacterial pathogens, including those associated with biological threats. TI antigens cannot be processed and presented to T cells and therefore are known to possess restricted T cell-dependent (TD) immunogenicity. Nevertheless, specific recognition of TI antigens by B1b cells and the highly protective antibody responses mounted by them clearly indicate a crucial role for this subset of B cells. Understanding the mechanisms of long-term immunity conferred by B1b cells may lead to improved vaccine efficacy for a variety of TI antigens