Analysis of Arg834Gln and Val902Glu type 2A von Willebrand disease mutations: studies with recombinant von Willebrand factor and correlation with patient characteristics

Type 2A von Willebrand disease (vWD), the most common qualitative form of vWD, is characterized by a relative decrease in circulating intermediate and high molecular weight (HMW) multimers. We studied the biosynthesis of recombinant von Willebrand factor (vWF) containing each of two type 2A vWD mutations previously reported by us, Arg834Gln and Val902Glu. The structure of recombinant Arg834Gln vWF within transfected COS-7 cells and the secretion of HMW multimers were similar to wild type vWF. The normal transport and secretion of Arg834Gln vWF, categorizes it as a group II type 2A mutation. In contrast, the Val90- 2Glu mutation resulted in intracellular proteolysis of vWF with the generation of a 176-kD fragment and retention of vWF between the endoplasmic reticulum and the Golgi complex. Moreover, the 176-kD fragment was also increased in plasma from patients with the Val902Glu mutation. Significantly impaired secretion and intracellular proteolysis of Val902Glu vWF categorizes a new sub-group of type 2A mutations. The intracellular proteolysis of vWF Val902Glu explains the lack of response to 1-deamino 8-D-arginine vasopressin (DDAVP) in patients who carry the mutation.     

Role of Toll‐like receptor 9 signaling on activation of nasal polyp–derived fibroblasts and its association with nasal polypogenesis

Background

Nasal polyposis is characterized by persistent inflammation and remodeling in sinonasal mucosa. Toll‐like receptor 9 (TLR9) is a DNA receptor of the innate immune system that plays a pivotal role in fibrosis and inflammatory responses. The aim of this study is to explore the expression, activity, and potential pathogenic role of TLR9 signaling in tissue remodeling in nasal polyp–derived fibroblasts (NPDFs).

Methods

Fibrotic and inflammatory responses elicited by type A CpG oligonucleotides were examined in the NPDFs by a combination of real‐time quantitative polymerase chain reaction, Western blot analysis, enzyme‐linked immunosorbent assay, and immunofluorescence staining. For these experiments, the NPDFs were stimulated with different TLR9 agonists (CpG A and B) and blocked with inhibitors (MyD88 inhibitor and chloroquine).

Results

TLR9 expression was significantly higher in nasal polyposis (NP) tissues compared to control or chronic rhinosinusitis (CRS) mucosa. In the NPDFs, TLR9 showed intracellular localization and expression of TLR9 was increased after treatment with CpG A. CpG A increased production of α‐smooth muscle actin (α‐SMA), fibronectin, and matrix metalloproteinases (MMPs) (MMP1, MMP2, and MMP9) in the NPDFs, while MyD88 inhibitor and chloroquine, which are known to block the TLR9 signaling pathway, inhibited their production. CpG A also produced type I interferons (IFN‐α and IFN‐β), which were inhibited by MyD88 inhibitor.

Conclusion

Our data indicates that CpG A–induced fibroblast activation and cytokine production were mediated via TLR9 stimulation in NPDFs. Disrupting this process with an inhibitor targeting TLR9 or its downstream signaling pathways could represent a novel approach to CRS with NP (CRSwNP) therapy.

     

The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts

Varying, limiting numbers of unseparated or purified cells (Ly-5.1), either from 14.5-day-old fetal liver (FL) or from adult bone marrow (BM) were coinjected with 10(5) unseparated BM cells (Ly-5.2) into lethally irradiated adult C57B1/6 recipients (Ly-5.2). The kinetics of donor cell repopulation of the lymphoid and myeloid compartments by Ly- 5.1+ donor hematopoietic stem cells (ie, competitive repopulation units [CRU]) were monitored at various time points after the transplantation by Ly-5 analysis of the peripheral white blood cells (WBC). Recipients that had received on average less than 2 adult BM or FL CRU did not show a significant difference in the level of donor-reconstitution when analyzed 4 weeks after the transplantation, However, at 8 and 16 weeks, the FL recipients showed a significantly higher percentage of donor- derived nucleated peripheral blood cells than did the recipients of adult BM cells. Analysis of individual mice showed that approximately 80% of the recipients of FL CRU showed an increase in mature WBC output between 4 and 8 weeks after transplantation, whereas this occurred in less than 40% in the recipients of adult BM cells. In addition to this effect on mature cell output, the cellularity of the reconstituted BM was significantly higher in recipients of FL CRU than in recipients of adult BM CRU, even at 7 to 9 months after transplantation, which is consistent with an increased clonal expansion of FL CRU. When marrow cells from primary recipients of FL CRU were injected into secondary recipients, a significantly higher percentage of these mice showed donor-reconstitution of their lymphoid and myeloid compartments (P < .01) and to a greater extent (P < .008) as compared with mice that had received marrow cells from primary recipients of similar numbers of adult BM CRU. Taken together, these results show that individual FL CRU exhibit a greater proliferative activity in vivo than similar cells from adult BM that is accompanied by a greater production of daughter CRU.     

Thrombocytopoietic properties of oncostatin M

Oncostatin M (OM) is a 28-kD glycoprotein that exhibits a panoply of biologic effects. Based on histologic observations of increased splenic megakaryocytes in nude mice implanted with an OM-secreting cell line, the thrombocytopoietic properties of OM in mice were investigated in culture and in vivo. Alone, OM did not induce megakaryocytic colony formation, but in combination with murine interleukin-3 (IL-3), OM markedly enhanced colony formation. The effects of OM on colony formation were similar to those of IL-6. OM alone augmented acetylcholinesterase in short-term marrow cultures. In normal mice, the administration of OM augmented platelet counts without increasing other circulating blood cell counts. The increment in counts exceeded that observed with IL-6. The kinetics of the OM response suggested that maximal increases in platelets occurred 3 days after the cessation of OM administration, irrespective of the duration of administration. In irradiated mice, OM administration accelerated platelet recovery and prevented the decrease in red blood cells observed in irradiated control animals. The data show that OM behaves as a megakaryocytic maturation factor in vitro and augments platelet production in vivo. Based on these animal data, OM may have potential clinical utility as a thrombocytopoietic agent.     

Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase

In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates.     

High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.     

Gastroparesis in patients with inactive Crohn's disease: a case series

Background  

Few studies have described patients with foregut dysmotility in inflammatory bowel disease. The aim of this case series was to evaluate clinical characteristics of 5 patients with inflammatory bowel disease and symptoms and signs of upper gut dysmotility.     

Cyclin D1 protein analysis in the diagnosis of mantle cell lymphoma

Mantle cell lymphoma (MCL) is a clinicopathologic entity that is difficult to diagnose on histopathologic criteria. Approximately 50% to 70% of MCL contain a t(11;14)(q13;q32) translocation involving the cyclin D1 gene. Irrespective of this rearrangement, almost all MCL show overexpression of the cyclin D1 gene at the mRNA level. Other B-cell non-Hodgkin's lymphomas (NHL) do not show this rearrangement or overexpression of cyclin D1. We developed an immunohistochemical assay to detect overexpression of the cyclin D1 protein on conventional formalin-fixed, paraffin-embedded biopsies using the well-defined monoclonal antibody DCS-6. Expression in tumor cells was compared with expression of cyclin D1 in endothelial cells and fibroblasts. An exclusively nuclear staining pattern was observed. Moreover, expression was directly compared with the expression observed by immunoblot analysis with the same antibody, as well as with mRNA expression and with the occurrence of genomic rearrangements within the BCL-1 locus. Of 13 MCL that were analyzed by immunohistochemistry and immunoblot, 12 showed overexpression with both techniques, whereas no overexpression was observed in 39 other NHL. Of 13 additional MCL studied either by immunohistochemistry or immunoblot, 11 also showed overexpression. Two lymphomas morphologically indistinguishable from MCL but with an aberrant immunophenotype (CD5 negative, CD10 positive) both lacked overexpression of cyclin D1. These results underscore the significance of overexpression of the cyclin D1 protein as a specific marker for MCL. Detection of cyclin D1 overexpression on formalin-fixed, paraffin- embedded tissues using the DCS-6 monoclonal antibody can be applied for routine diagnostic purposes.     

Production of genetically stable high-titer retroviral vectors that carry a human gamma-globin gene under the control of the alpha-globin locus control region

The ability to generate stable high-titer vectors that give rise to high levels of expression of transduced globin genes in erythroid cells is a prerequisite for effective retroviral-mediated globin gene therapy. The human beta-globin gene with its immediate flanking sequences does not contain all the regulatory elements necessary for regulated high-level and position-independent expression in erythroid cells. The regulatory element known as the beta-globin locus control region (BetaLCR) can provide a linked Beta-globin gene with these properties. However, addition of BetaLCR sequences to a retrovirus carrying a beta-globin gene increases its genetic instability. We have developed a new generation of retroviral vectors in which a human gamma- globin gene is placed under the control of the alphaLCR, the major regulatory element of the alpha-globin gene cluster. We demonstrate that these retroviruses are genetically stable in producer cell lines and can be produced at high titers that exceed 5 x 10(6) colony-forming units (CFU)/mL. In addition, we show that the transduced gamma-globin gene can be expressed in the adult erythroid environment of mouse erythroleukemia (MEL) cells at a level comparable to that of a single endogenous Betamaj-globin gene. These retroviruses can also transduce primary murine bone marrow progenitor cells as efficiently as retroviruses that carry the neomycin resistance (neor) gene. This new generation of globin retroviral vectors may prove useful for gene therapy of human beta-globin gene disorders such as sickle cell disease and beta-thalassemia.     

Bone marrow transplantation for patients with Philadelphia chromosome- positive acute lymphoblastic leukemia

We report the treatment outcome of allogeneic bone marrow transplantation in ten patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. Six patients are alive and well for 6 to 30 months (median 19 months) after transplantation. Four patients died with transplant related complications. In view of the poor prognosis associated with this disease, marrow ablation followed by allogeneic or syngeneic marrow grafting may be the preferred treatment modality if a suitable marrow donor is available.