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

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 kappa) 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 V(H)3-64 in the germ line configuration. G14 and G19 use V(H)6-1, which has somatic mutations. All of the MAbs use V kappa 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 V(H) gene use may influence GXM specificity and efficacy, and they provide insights into the possible contribution that V(H) gene use may have in resistance and susceptibility to cryptococcosis.     

Absolute immature platelet count helps differentiate thrombotic thrombocytopenic purpura from hypertension-induced thrombotic microangiopathy

ADAMTS13 activity measurement is used in the diagnostic algorithm of thrombotic thrombocytopenic purpura (TTP), but results may not be available before initiation of therapeutic plasma exchange (TPE). The immature platelet fraction (%-IPF) and the calculated absolute immature platelet count (A-IPC) represent a test of real-time thrombopoiesis, and can be performed in most laboratories using automated analyzers. Here we report on using A-IPC kinetics to exclude idiopathic TTP in a patient with severe hypertension, thrombocytopenia, and acute renal failure, which was confirmed by a normal ADAMTS13. The complete resolution of thrombocytopenia occurred once blood pressure was controlled favoring a diagnosis of hypertension-induced thrombotic microangiopathy.     

Comparison of BIOMED-2 Versus Laboratory-Developed Polymerase Chain Reaction Assays for Detecting T-Cell Receptor-γ Gene Rearrangements

Detecting clonal T-cell receptor (TCR)-γ gene rearrangements (GRs) is an important adjunct test for diagnosing T-cell lymphoma. We compared a recently described assay (BIOMED-2 protocol), which targets multiple variable (V) gene segments in two polymerase chain reaction (PCR) reactions (multi-V), with a frequently referenced assay that targets a single V gene segment in four separate PCR reactions (mono-V). A total of 144 consecutive clinical DNA samples were prospectively tested for T-cell clonality by PCR using laboratory-developed mono-V and commercial multi-V primer sets for TCR-γ GR. The combination of TCR-β, mono-V TCR-γ and multi-V TCR-γ detected more clonal cases (68/144, 47%) than any individual PCR assay. We detected clonal TCR-β GR in 47/68 (69%) cases. Using either mono-V or multi-V TCR-γ primers, the sensitivities for detecting clonality were 52/68 (76%) or 51/68 (75%). Using both mono-V and multi-V TCR-γ primers improved the sensitivity for detecting clonality, 60/68 (88%). Combining either mono-V or multi-V TCR-γ primers with TCR-β primers also improved the sensitivity, 64/68 (94%). Significantly, TCR-γ V11 GRs could only be detected using the mono-V-PCR primers. We conclude that using more than one T-cell PCR assay can enhance the overall sensitivity for detecting T-cell clonality.The ability to distinguish benign from malignant T-cell proliferations relies on a combination of morphological, immunological and molecular tests. Often a clonal T-cell receptor (TCR) gene rearrangement (GR) is the only definitive marker for neoplasia. While Southern blot analysis is the gold standard for detecting antigen receptor GRs, it is not suitable for small biopsies or formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, most laboratories depend on the polymerase chain reaction (PCR) for detecting TCR GRs. Because of the complexity of the TCR-β locus,¹ routine testing has focused on the TCR-γ locus,^{2,3,4,5,6,7,8,9,10,11,12} which contains fewer variable (V) gene segments.¹ Even though TCR-γ comprises only 11 functional V regions, several different PCR strategies are in use.^13,14We rigorously compared the recently described BIOMED-2 TCR-γ assay, which targets two different TCR-γ V gene segments (V1-8/V10 and V9/V11) in two separate reactions (multi-V strategy; Figure 1A)¹⁵ against a previously described TCR-γ laboratory-developed assay, which has been cited more than 100 times (ISI Web of knowledge citation index; Thompson Reuters, Philadelphia, PA), which targets a single TCR-γ V gene segment (V1-8, V9, V10, V11) in each of four individual PCR reactions (mono-V strategy; Figure 1A).¹⁶ In a sense, both TCR-γ PCR strategies can be considered variants of multiplex strategies because they combine one or two V primers with multiple J primers in each PCR reaction:^15,16 the multi-V strategy adds the JP1/JP2 and J1/J2 primers¹⁵ and the mono-V strategy adds the JP1/JP2, J1/J2 and JP primers¹⁶ (Figure 1B). It has been suggested that because of possible competition between amplicons for reagents and interactions between primers, multiplex PCR may be more susceptible to reduced sensitivity.^17,18 Therefore, we compared the mono-V versus the multi-V PCR strategies for detecting TCR-γ gene rearrangements in a large number of clinical DNA samples.Open in a separate windowFigure 1A: Schematic representation of the TCR-γ genomic DNA locus. Approximate positions of the primers for both mono-V and multi-V PCR strategies are marked by arrows. The bars represent consensus primer-binding sequences. * ← JP primer is only used in the mono-V assay. B:TCR-γ variable gene segments V1-8, V9, V10, V11 (forward primers) and J segments (reverse primers): Reference DNA sequences from GenBank (top), mono-V TCR-γ primers (middle), multi-V TCR-γ primers (bottom). All DNA sequences are in 5′ to 3′ orientation. C: Detection of TCR-γ PCR products by capillary gel electrophoresis. Mono-V (left column) and multi-V (right column) PCR assays amplified representative DNA samples from the same patient.     

Emergency ABO‐incompatible liver transplant secondary to fulminant hepatic failure: Outcome,role of TPE and review of the literature

The increasing demand for solid organ transplants has brought to light the need to utilize organs in critical situations despite ABO‐incompatibility. However, these transplantations are complicated by pre‐existing ABO antibodies which may be potentially dangerous and makes the transplantation prone to failure due to rejection with resulting necrosis or intrahepatic biliary complications. We report the clinical outcome of an emergency ABO‐incompatible liver transplant (due to fulminant hepatic failure with sudden and rapidly deteriorating mental status) using a modified therapeutic plasma exchange (TPE) protocol. The recipient was O‐positive with an initial anti‐B titer of 64 and the cadaveric organ was from a B‐positive donor. The patient underwent initial TPE during the peri‐operative period, followed by a series of postoperative daily TPE, and later a third series of TPE for presumptive antibody‐mediated rejection. The latter two were performed in conjunction with the use of IVIg and rituximab. The recipient's anti‐B titer was reduced and maintained at 8 or less 8 months post‐op. However, an elevation of transaminases 3 months post‐transplant triggered a biopsy which was consistent with cellular rejection and with weak C4d positive staining suggestive of antibody mediated rejection. Additional plasma exchange procedures were performed. The patient improved rapidly after modification of her immunosuppression regimen and treatment with plasma exchange. This case illustrates that prompt and aggressive plasma exchange, in conjunction with immunosuppression, is a viable approach to prevent and treat antibody mediated transplant rejection in emergency ABO‐incompatible liver transplant. J. Clin. Apheresis, 2012. © 2012 Wiley Periodicals, Inc.     

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. α-synuclein is a major component of the Lewy body of neurodegenerative diseases and γ-synuclein is a marker of aggressive carcinomas. As the role of γ-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-γ-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 γ-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; γ-synuclein highlighted both the long slender cellular processes and the cell body, thereby clearly demonstrating the number of individual FDCs. In addition, γ-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 γ-synuclein; however, γ-synuclein was not expressed by 11 other types of SSNs tested. These results suggest that γ-synuclein is a promising new adjunct marker for identifying reactive FDCs and for diagnosing FDC sarcoma and benign and malignant vascular tumors.     

Absolute immature platelet counts in the setting of suspected heparin-induced thrombocytopenia may predict anti-PF4-heparin immunoassay testing results

Background

Heparin-induced-thrombocytopenia (HIT) is a disease mediated by antibodies to platelet factor 4 (PF4)-heparin complexes. Immature platelet fraction (%-IPF) and absolute immature platelet count (A-IPC) measure newly-released platelets into circulation and can prove useful in differentiating patients with thrombocytopenic presentations due to consumptive or hypoproduction processes. Therefore, we evaluated utility of A-IPC in a cohort of thrombocytopenic patients suspected of HIT.

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.