Robert W. Maitta

Immunogenicity and Efficacy of Cryptococcus neoformans Capsular Polysaccharide Glucuronoxylomannan Peptide Mimotope-Protein Conjugates in Human Immunoglobulin Transgenic Mice

ABSTRACT Peptide mimotopes of capsular polysaccharides have been proposed as antigens for vaccines against encapsulated pathogens. In this study, we determined the antibody response to and efficacy of P13, a peptide mimetic of the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM), in mice that produce human antibodies. P13 was conjugated to tetanus toxoid (TT) or diphtheria toxoid (DT) and administered subcutaneously in Alhydrogel with or without CpG to mice transgenic for human immunoglobulin loci (XenoMouse mice) and expressing either immunoglobulin G2 (IgG2) (G2 mice) or IgG4 (G4 mice). Mice were vaccinated and revaccinated two or three times. The serum antibody responses of the mice to GXM and P13 and antibody idiotype expression were analyzed by an enzyme-linked immunosorbent assay. The results showed that both P13-TT and P13-DT were antigenic, inducing a mimetic response to P13 in both G2 and G4 mice, and immunogenic, inducing a mimotope response including VH3 (idiotype)-positive antibodies to GXM in G2 but not G4 mice. CpG led to higher titers of IgG to P13 and GXM in P13-TT-vaccinated G2 mice. C. neoformans challenge of P13-protein conjugate-vaccinated and control G2 mice induced anamnestic IgG- and VH3-positive responses to GXM and was associated with a significantly decreased risk of death and a prolongation of survival in P13-DT-vaccinated mice compared to phosphate-buffered saline-treated or protein carrier-vaccinated mice. These findings reveal that P13 elicited a human antibody response with VH3 expression in human immunoglobulin transgenic mice that has been observed for human antibodies to GXM and support the concept that peptide mimotope-based vaccines may hold promise for the treatment of C. neoformans infections.

Detection of septic transfusion reactions to platelet transfusions by active and passive surveillance

Septic transfusion reactions (STRs) resulting from transfusion of bacterially contaminated platelets are a major hazard of platelet transfusion despite recent interventions. Active and passive surveillance for bacterially contaminated platelets was performed over 7 years (2007-2013) by culture of platelet aliquots at time of transfusion and review of reported transfusion reactions. All platelet units had been cultured 24 hours after collection and released as negative. Five sets of STR criteria were evaluated, including recent AABB criteria; sensitivity and specificity of these criteria, as well as detection by active and passive surveillance, were determined. Twenty of 51,440 platelet units transfused (0.004%; 389 per million) were bacterially contaminated by active surveillance and resulted in 5 STRs occurring 9 to 24 hours posttransfusion; none of these STRs had been reported by passive surveillance. STR occurred only in neutropenic patients transfused with high bacterial loads. A total of 284 transfusion reactions (0.55%) were reported by passive surveillance. None of these patients had received contaminated platelets. However, 6 to 93 (2.1%-32.7%) of these 284 reactions met 1 or more STR criteria, and sensitivity of STR criteria varied from 5.1% to 45.5%. These results document the continued occurrence of bacterial contamination of platelets resulting in STR in neutropenic patients, failure of passive surveillance to detect STR, and lack of specificity of STR criteria. These findings highlight the limitations of reported national STR data based on passive surveillance and the need to implement further measures to address this problem such as secondary testing or use of pathogen reduction technologies.

Protective and nonprotective human immunoglobulin M monoclonal antibodies to Cryptococcus neoformans glucuronoxylomannan manifest different specificities and gene use profiles

ABSTRACT The features of protective murine antibodies to the Cryptococcus neoformans capsular polysaccharide glucuronoxylomannan (GXM) have been rigorously investigated; however, the characteristics of protective human antibodies to GXM have not been defined. We produced monoclonal antibodies (MAbs) from XenoMouse mice (transgenic mice that express human immunoglobulin M [IgM], IgG2, and κ) which were immunized with a C. neoformans serotype D strain 24067 GXM-diphtheria toxoid conjugate. This study reports the specificity and efficacy of three human IgM MAbs, G14, G15, and G19, generated from these mice. Each MAb was specific for GXM, but G14 and G19 had different specificity based on their binding to serotype A strain H99 and SB4 GXMs, to which G15 did not bind. Nucleic acid sequence analysis revealed that G15 uses VH3-64 in the germ line configuration. G14 and G19 use VH6-1, which has somatic mutations. All of the MAbs use Vκ DPK22/A27. Studies of MAb efficacy in BALB/c mice showed that administration of 0.1 mg, but not 1 or 0.01 mg, of G15 prolonged survival against lethal C. neoformans strain 24067 challenge, whereas G14 and G19 were not protective at any dose. This panel of MAbs illustrates that serotype D GXM has epitopes that elicit human antibodies that can be either protective or nonprotective. Our findings suggest that VH gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that VH gene use may have in resistance and susceptibility to cryptococcosis.

Late stages of hematopoiesis and B cell lymphopoiesis are regulated by α-synuclein, a key player in Parkinson's disease.

α-Synuclein plays a crucial role in Parkinsons disease and dementias defined as synucleinopathies. α-Synuclein is expressed in hematopoietic and immune cells, but its functions in hematopoiesis and immune responses are unknown. We utilized α-synuclein(-/-) (KO) mice to investigate its role in hematopoiesis and B cell lymphopoiesis. We demonstrated hematologic abnormalities including mild anemia, smaller platelets, lymphopenia but relatively normal early hematopoiesis in KO mice compared to wild-type (WT) as measured in hematopoietic stem cells and progenitors of the different cell lineages. However, the absolute number of B220(+)IgM(+) B cells in bone marrow was reduced by 4-fold in KO mice (WT: 104±23×10(5) vs. KO: 27±5×10(5)). B cells were also reduced in KO spleens associated with effacement of splenic and lymph node architecture. KO mice showed reduced total serum IgG but no abnormality in serum IgM was noted. When KO mice were challenged with a T cell-dependent antigen, production of antigen specific IgG1 and IgG2b was abolished, but antigen specific IgM was not different from WT mice. Our study shows hematologic abnormalities including anemia and smaller platelets, reduced B cell lymphopoiesis and defects in IgG production in the absence of α-synuclein. This is the first report to show an important role of α-synuclein late in hematopoiesis, B cell lymphopoiesis and adaptive immune response.

A critical role for alpha-synuclein in development and function of T lymphocytes

Alpha-synuclein is highly expressed in the central nervous system and plays an important role in pathogenesis of neurodegenerative disorders such as Parkinsons disease and Lewy body dementia. Previous studies have demonstrated the expression of α-synuclein in hematopoietic elements and peripheral blood mononuclear cells, although its roles in hematopoiesis and adaptive immunity are not studied. Using an α-synuclein knock out (KO) mouse model, we have recently shown that α-synuclein deficiency is associated with a mild defect in late stages of hematopoiesis. More importantly, we demonstrated a marked defect in B lymphocyte development and IgG, but not IgM production in these mice. Here we show a marked defect in development of T lymphocytes in α-synuclein KO mice demonstrated by a significant increase in the number of CD4 and CD8 double negative thymocytes and significant decreases in the number of CD4 single positive and CD8 single positive T cells. This resulted in markedly reduced peripheral T lymphocytes. Interestingly, splenic CD4(+) and CD8(+) T cells that developed in α-synuclein KO mice had a hyperactivated state with higher expression of early activation markers and increased IL-2 production. Moreover, splenic CD4(+) T cells from α-synuclein KO mice produced lower levels of IL-4 upon antigenic stimulation suggesting a defective Th2 differentiation. Our data demonstrate an important role for α-synuclein in development of T lymphocytes and regulation of their phenotype and function.

The safety of the blood supply-time to raise the bar

To the Editor: In their Perspective article on pathogen-reduction technology (PRT) (May 14 issue),1 Snyder et al. praise the value of this technology. However, they omit a discussion of concerns about the safety and efficacy of PRT, stating that “Numerous studies demonstrate little substantive negative effect from pathogen reduction on plasma proteins or platelets.” A study in the Netherlands was terminated because of increased bleeding complications in the PRT group,2 and a U.S. study documented five fatal cases of the acute respiratory distress syndrome in the PRT group (1.6%) versus none in the control group.3 Furthermore, PRT fails to inactivate some pathogens, such as the hepatitis E virus. Among bacterial pathogens, PRT was not 100% effective against high levels of certain Klebsiella pneumoniae strains and spore-forming Bacillus cereus.4 A further challenge to PRT is its high cost. The use of “at-issue” tests that are approved by the Food and Drug Administration (FDA) to detect bacterial contamination, one of which has been shown to be highly effective in a clinical study,5 is not promoted. The potential for PRT to improve safety and prevent the transmission of infection needs to be tempered by these considerations.

Association of MLL amplification with poor outcome in acute myeloid leukemia.

Chromosomal rearrangements and amplification of the MLL gene at 11q23 are common abnormalities found in patients with severe myelodysplastic disorders and lymphoid and acute myeloid leukemias. MLL rearrangements are associated with aggressive disease in both children and adults, with current evidence suggesting that MLL alterations are associated with a poor prognosis. We report the clinical, cytogenetic and histologic findings of a patient who presented with a de novo diagnosis of AML-M4 and who fits the profile of patients presenting with MLL alterations, such as old age at presentation, rapid progression, therapeutic refractoriness, and poor outcome. Two bone marrow specimens taken 1 month apart show the rapid deterioration of the patients cytogenetic abnormalities at the 11q23 locus, with amplification of MLL that was originally seen as a homogeneously staining region (hsr) on chromosome 11. In the second biopsy the hsr and MLL amplification appears as nonreciprocal translocation of multiple copies in the form of marked amplification of MLL on chromosome 16 in a background of increasing chromosomal aberrations. This case suggests that either the MLL amplification and translocation alone or in conjunction with other flanking oncogenes may have played an important role in poor patient outcome.

Absolute immature platelet count dynamics in diagnosing and monitoring the clinical course of thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a life‐threatening diagnosis requiring prompt initiation of therapeutic plasma exchange (TPE). Measurement of immature platelet (PLT) fraction (%‐IPF) differentiates PLT consumption or destruction from hypoproduction.

γ-Synuclein is a promising new marker for staining reactive follicular dendritic cells, follicular dendritic cell sarcoma, Kaposi sarcoma, and benign and malignant vascular tumors.

Synucleins are small soluble proteins found in normal brain that facilitate rapid release of neurotransmitters. &agr;-synuclein is a major component of the Lewy body of neurodegenerative diseases and &ggr;-synuclein is a marker of aggressive carcinomas. As the role of &ggr;-synuclein has not yet been investigated in the lymphoid system, we immunohistochemically stained normal lymphoid organs, lymph nodes with reactive lymphoid hyperplasia, and malignant lymphomas. The anti-&ggr;-synuclein antibody strongly stained the follicular dendritic cell (FDC) meshworks and vascular and lymphatic endothelial cells in reactive lymphoid tissues, in B-cell lymphomas with a nodular pattern, and in angioimmunoblastic T-cell lymphomas. There were no &ggr;-synuclein-positive FDC meshworks in B-cell or T-cell lymphomas with a diffuse pattern. This is in contrast to CD21, which only stained the arms of the FDCs; &ggr;-synuclein highlighted both the long slender cellular processes and the cell body, thereby clearly demonstrating the number of individual FDCs. In addition, &ggr;-synuclein was strongly expressed by the neoplastic counterpart of reactive FDCs (FDC sarcoma) and by the neoplastic counterparts of normal lymphatic and vascular endothelial cells (Kaposi sarcoma, hemangioma, and angiosarcoma). Only a few spindle cell neoplasms (SSNs) derived from smooth muscle, peripheral nerve, or gastrointestinal stroma expressed &ggr;-synuclein; however, &ggr;-synuclein was not expressed by 11 other types of SSNs tested. These results suggest that &ggr;-synuclein is a promising new adjunct marker for identifying reactive FDCs and for diagnosing FDC sarcoma and benign and malignant vascular tumors.

Alpha- and beta-synucleins are new diagnostic tools for acute erythroid leukemia and acute megakaryoblastic leukemia.

α-Synuclein is a key component of the Lewy body, a large globular protein complex that forms in the nervous system of patients with Parkinson disease and other dementias [1-3]. Since α-synuclein also occurs in megakaryocytic and erythroid lineages [4-7], we wondered what role synucleins had in the hematopoietic system. Therefore, we studied the expression of α-, β-, and γ-synucleins in a comprehensive panel of patient bone marrows and leukemic cell lines. We observed under expression of α-synuclein in the megakaryocytes of myeloproliferative neoplasm (MPN), but not normal reactive marrow (NRM) or myelodysplastic syndrome (MDS). Conversely, we observed over expression of β-synuclein in the blasts of megakaryoblastic leukemias (MegL), but not acute myeloid leukemia (AML) or erythroleukemia (EryL), suggesting that α- and β-synucleins could be useful adjunct markers for the early detection of MDS and the differential diagnosis of EryL and MegL from other AMLs.