Minor histocompatibility antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T-cell clones inhibit human hematopoietic progenitor cell growth by a mechanism that is dependent on direct cell-cell contact

HLA-identical bone marrow transplantation (BMT) may be complicated by graft-versus-host disease or graft rejection. Both complications are thought to be initiated by recognition of minor histocompatibility (mH) antigens by HLA-restricted mH-antigen-specific T lymphocytes. Using HLA- A2-restricted mH antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T lymphocyte (CTL) clones, we studied the recognition by these CTL clones of interleukin-2 (IL-2)-stimulated T cells (IL-2 blasts), BM mononuclear cells (BMMNCs), and hematopoietic progenitor cells (HPCs). We showed that, when IL-2 blasts from the BM donors who were investigated were recognized by the HA-1-, -2-, and -4-, and HY- specific CTL clones, their BMMNCs and HPCs were recognized as well by these CTL clones, resulting in antigen-specific growth inhibition of erythrocyte burst-forming units (BFU-E), colony-forming units- granulocyte (CFU-G), and CFU-macrophage (CFU-M). the HA-2-specific CTL clone, however, inhibited BFU-E and CFU-G growth from four donors to a lesser extent than from two other donors. We further investigated whether inhibitory cytokines released into the culture medium by the antigen-specific stimulated CTLs or by stimulated BMMNCs were responsible for suppression of HPC growth or whether this effect was caused by direct cell-cell contact between CTLs and HPCs. HPC growth inhibition was only observed after preincubation of BMMNCs and CTLs together for 4 hours before plating the cells in semisolid HPC culture medium. When no cell-cell contact was permitted before plating, neither antigen-stimulated CTL nor antigen-nonstimulated CTLs provoked HPC growth inhibition. Culturing BMMNCs in the presence of supernatants harvested after incubation of BMMNCs and CTL clones together for 4 or 72 hours did also not result in HPC growth inhibition. Both suppression of HPC growth and lysis of IL-2 blasts and BMMNCs in the 51Cr-release assay appeared to be dependent on direct cell-cell contact between target cells and CTLs and were not caused by the release of inhibitory cytokines into the culture medium by antigen-specific stimulated CTLs or by stimulated BMMNCs. Our results show that mH-antigen-specific CTLs can inhibit HPC growth by a direct cytolytic effect and may therefore be responsible for BM graft rejection after HLA-identical BMT.     

Immunohistochemical localization of membrane and alpha-granule proteins in human megakaryocytes: application to plastic-embedded bone marrow biopsy specimens

Using a new technique for antigen localization, we have demonstrated platelet proteins in megakaryocytes in plastic-embedded biopsy specimens of normal human bone marrow. In a series of 25 specimens, megakaryocytes showed labeling with antibodies to the integral membrane glycoproteins IIIa, IIb, and the IIb-IIIa complex; granule membrane protein 140; and five alpha-granule matrix proteins: thrombospondin, factor VIII-related antigen, beta-thromboglobulin, platelet factor 4, and fibrinogen. The antibodies to the membrane glycoproteins IIIa, IIb, and IIb-IIIa produced diffuse cytoplasmic staining and heavier staining on the plasma membrane, whereas the antibodies to the alpha-granule matrix proteins produced a distinct granular staining within the cytoplasm. Staining for granule membrane protein 140 was also granular in distribution. Rare mononuclear cells consistent with megakaryocyte precursors were labeled with these markers. Other enzyme histochemical and lectin-binding studies showed that the enzyme alpha-naphthyl acetate esterase, the lectin Ulex europaeus I, and the periodic-acid Schiff reaction were consistent, but not specific, markers of megakaryocytes. This immunohistochemical technique should facilitate the examination of qualitative and quantitative changes in megakaryocytes in a variety of physiologic and pathologic processes.     

Activated protein C resistance: molecular mechanisms based on studies using purified Gln506-factor V

Gln506-factor V (FV) was purified from plasma of an individual homozygous for an Arg506Gln mutation in FV that is associated with activated protein C (APC) resistance. Purified Gln506-FV, as well as Gln506-FVa generated by either thrombin or FXa, conveyed APC resistance to FV-deficient plasma in coagulation assays. Clotting assay studies also suggested that APC resistance does not involve any abnormality in FV-APC-cofactor activity. In purified reaction mixtures, Gln506-FVa in comparison to normal FVa showed reduced susceptibility to APC, because it was inactivated approximately 10-fold slower than normal Arg506-FVa. It was previously reported that inactivation of normal FVa by APC involves an initial cleavage at Arg506 followed by phospholipid- dependent cleavage at Arg306. Immunoblot and amino acid sequence analyses showed that the 102-kD heavy chain of Gln506-FVa was cleaved at Arg306 during inactivation by APC in a phospholipid-dependent reaction. This reduced but measurable susceptibility of Gln506-FVa to APC inactivation may help explain why APC resistance is a mild risk factor for thrombosis because APC can inactivate both normal FVa and variant Gln506-FVa. In summary, this study shows that purified Gln506- FV can account for APC resistance of plasma because Gln506-FVa, whether generated by thrombin or FXa, is relatively resistant to APC.     

Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin

The hemagglutinin (HA) of influenza A(H3N2) virus responsible for the 1968 influenza pandemic derived from an avian virus. On introduction into humans, its receptor binding properties had changed from a preference for avian receptors (α2,3-linked sialic acid) to a preference for human receptors (α2,6-linked sialic acid). By 2001, the avidity of human H3 viruses for avian receptors had declined, and since then the affinity for human receptors has also decreased significantly. These changes in receptor binding, which correlate with increased difficulties in virus propagation in vitro and in antigenic analysis, have been assessed by virus hemagglutination of erythrocytes from different species and quantified by measuring virus binding to receptor analogs using surface biolayer interferometry. Crystal structures of HA–receptor analog complexes formed with HAs from viruses isolated in 2004 and 2005 reveal significant differences in the conformation of the 220-loop of HA1, relative to the 1968 structure, resulting in altered interactions between the HA and the receptor analog that explain the changes in receptor affinity. Site-specific mutagenesis shows the HA1 Asp-225→Asn substitution to be the key determinant of the decreased receptor binding in viruses circulating since 2005. Our results indicate that the evolution of human influenza A(H3N2) viruses since 1968 has produced a virus with a low propensity to bind human receptor analogs, and this loss of avidity correlates with the marked reduction in A(H3N2) virus disease impact in the last 10 y.Surveillance of influenza viruses is essential for updating vaccines, for tracking the emergence of drug resistant viruses, and for monitoring zoonotic infections. It also gives important insights into the mechanisms of virus evolution. This is particularly the case for interpreting the correlation between antigenic differences and changes in the sialic acid receptor binding properties of the HA glycoprotein. The correlation in these two properties arises because of the close proximity on HA of binding sites for antibodies that neutralize virus infectivity and the sialic acid receptor binding pocket (1), and accounts for the observations that mutations that prevent antibody binding can also result in changes in receptor binding (2–7). Reduction in affinity of human H3N2 viruses for avian receptors since the beginning of the pandemic in 1968 has meant that by the 1990s viruses with reduced ability to agglutinate chicken erythrocytes had emerged (8, 9). Moreover, viruses isolated after 1999 were shown to have reduced affinity for both human and avian receptors, a feature that correlated with their poor growth properties in eggs and different cells in culture (9–14). The evolution of the HA has resulted in at least three key changes that influence receptor binding. Two sequential substitutions occurred at residue 225: in 2001–2002, a substitution Gly-225→Asp was accompanied by a Trp-222→Arg substitution, and in 2004–2005, an Asp-225→Asn substitution was accompanied by the substitution Ser-193→Phe (while maintaining arginine at position 222). Residue 226, a key amino acid in determining receptor specificity (15), also changed twice: before 2001, Leu-226→Val, and in 2004, Val-226→Ile (Fig. S1).To correlate these amino acid substitutions with the biological properties of the viruses, we have analyzed the receptor binding characteristics of H3N2 viruses isolated between 2001 and 2010, examined changes in their ability to infect cells in culture, and determined the structures of two HAs of virus isolates from 2004 and 2005 in the absence of receptor and complexed with a human receptor analog. The data show that the progressive decrease in binding of these viruses to human receptors from 2000 onward correlates with changes in the efficiencies of infection of cultured cells. Comparison of structural data for HAs of viruses from 1968, 2004, and 2005 explain how particular mutations that affect the conformation of the HA1 220-loop component of the receptor binding site define the receptor binding phenotype of recent H3N2 human influenza viruses.     

Laser-induced thermal stress and the heat shock response in neural cells

Background?The Ho: YAG laser is used extensively in orthopedic surgery. It offers a minimally invasive method of ablating tissue with precision. Previous studies have explored the effects of laser use on temperature during experimental foraminoplasty. To date, there has been limited work on the effects of thermal stress on cells in this context.Material and methods?Cells were exposed either to heated medium or the Ho: YAG laser in the high-power mode. Heated medium was used as a stressor by (I) exposing groups of cells to a constant temperature of 45°C for varying lengths of time: 5, 10, 15 and 20 min, and (II) exposing cells for a fixed length of time (5 min) to varying temperatures: 45°C, 55°C, 65°C with a control treated at 37°C. A third group was subjected to direct laser treatment. The effects of the treatments were assessed using trypan blue staining as a measure of viability and immunocytochemistry was used to measure changes in heat shock protein (HSP) expression.Results?There was a negative correlation between cell viability and HSP expression, and between cell viability and the severity of the treatment.Interpretation?Our findings suggest a possible role for the Ho: YAG laser in spinal foraminoplasty based on the high level of cell viability in the treatment regimen that most closely mirrored the clinical application of the laser.     

Surgical delivery of drug releasing poly(lactic-co-glycolic acid)/poly(ethylene glycol) paste with in vivo effects against glioblastoma

Introduction

The median survival of patients with glioblastoma multiforme (astrocytoma grade 4) remains less than 18 months despite radical surgery, radiotherapy and systemic chemotherapy. Surgical implantation of chemotherapy eluting wafers into the resection cavity has been shown to improve length of survival but the current licensed therapy has several drawbacks. This paper investigates in vivo efficacy of a novel drug eluting paste in glioblastoma.

Methods

Poly(lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) self-sintering paste was loaded with the chemotherapeutic agent etoposide and delivered surgically into partially resected tumours in a flank murine glioblastoma xenograft model.

Results

Surgical delivery of the paste was successful and practical, with no toxicity or surgical morbidity to the animals. The paste was retained in the tumour cavity, and preliminary results suggest a useful antitumour and antiangiogenic effect, particularly at higher doses. Bioluminescent imaging was not affected significantly by the presence of the paste in the tumour.

Conclusions

Chemotherapy loaded PLGA/PEG paste seems to be a promising technology capable of delivering active drugs into partially resected tumours. The preliminary results of this study suggest efficacy with no toxicity and will lead to larger scale efficacy studies in orthotopic glioblastoma models.