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Peripheral blood mononuclear cells (PBMCs) are the only source of human lymphoid cells routinely available for immunomonitoring of T-cell responses to microbial and tumor-associated antigens. However, previous work in mice and humans had indicated that CD4 T cells transiently lose antigen sensitivity when cellular contacts are lost (eg, by entering the circulation). Using the simple and robust protocol for resetting T cells to original reactivity (RESTORE; ie, preculturing PBMCs for 2 days at a high cell density before initiation of antigenic stimulation), we show that CD8 T-cell responses to viral and tumor-associated antigens are greatly underestimated in blood, and sometimes even remain undetected, if conventional, unprocessed PBMC cultures are used. The latter finding is particularly striking with regard to the appearance of Wilms tumor 1 protein-specific CD8 T-cell responses in leukemia patients after allogeneic bone marrow transplantation. The dramatic increase in antigen sensitivity of "restored" CD8 T cells is associated with phosphorylation of proximal T-cell receptor signaling components, and with the upregulation of genes involved in aerobic glycolysis, thereby increasing T-cell functionality. The RESTORE protocol permits a more meaningful monitoring of CD8 memory T-cell responses to viral infections and tumors and vaccination success. Furthermore, when generating T-cell lines for adoptive T-cell therapy, it avoids the loss of those clones, which strictly depend on the primed status conferred by cellular interactions in the tissue context for their initial reactivation by antigen. The data reported in this article have been deposited in the Gene Expression Omnibus database (accession number GSE63430)., (© 2015 by The American Society of Hematology.)

Intrinsic and extrinsic apoptotic pathways converge to activate common downstream executioner caspases (caspase-3, -6, and -7), resulting in cell death. In chronic lymphocytic leukemia (CLL), neoplastic B cells evade apoptosis owing to the overexpression of survival proteins. We hypothesized that direct activation of procaspases could bypass the apoptosis resistance induced by the upstream prosurvival proteins. The procaspase-activating compounds (PAC-1), including B-PAC-1 (L14R8), convert inactive executioner procaspases to their active cleaved forms by chelation of labile zinc ions. Both at transcript and protein levels, primary CLL cells express high levels of latent procaspases (3, -7, and -9). B-PAC-1 treatment induced CLL lymphocyte death which was higher than that in normal peripheral blood mononuclear cells or B cells, and was independent of prognostic markers and microenvironmental factors. Mechanistically, B-PAC-1 treatment activated executioner procaspases and not other Zn-dependent enzymes. Exogenous zinc completely, and pancaspase inhibitors partially, reversed B-PAC-1-induced apoptosis, elucidating the zinc-mediated mechanism of action. The cell demise relied on the presence of caspase-3/7 but not caspase-8 or Bax/Bak proteins. B-PAC-1 in combination with an inhibitor of apoptosis protein antagonist (Smac066) synergistically induced apoptosis in CLL samples. Our investigations demonstrated that direct activation of executioner procaspases via B-PAC-1 treatment bypasses apoptosis resistance and is a novel approach for CLL therapeutics., (© 2015 by The American Society of Hematology.)

Several peripheral blood mononuclear cell (PBMC)-derived cell populations can promote angiogenesis, and differences in CD34(+) or CD14(+) surface expression have been used to separate PBMC subpopulations in this respect. AngiomiRs, microRNAs regulating angiogenesis, are key regulators of angiogenic processes. The present study examines differential angiomiR expression/secretion from CD34(+)/CD14(+), CD34(+)/CD14(-), CD34(-)/CD14(+), and CD34(-)/CD14(-) PBMC subsets and their relevance for different proangiogenic properties. Notably, both circulating human CD34(+)/14(+) and CD34(+)/14(-) PBMC subsets and their supernatants exerted more potent proangiogenic effects compared with CD34(-) PBMC subsets. MiR-126 was identified as most differentially expressed angiomiR in CD34(+) compared with CD34(-) PBMC subsets, determined by miR-array and RT-PCR validation. Modulation of miR-126 by anti-miR-126 or miR-mimic-126 treatment resulted in significant loss or increase of proangiogenic effects of CD34(+) PBMCs. MiR-126 levels in supernatants of CD34(+) PBMC subsets were substantially higher compared with CD34(-) PBMC subsets. MiR-126 was secreted in microvesicles/exosomes, and inhibition of their release impaired CD34(+) PBMCs proangiogenic effects. Notably, high-glucose treatment or diabetes reduced miR-126 levels of CD34(+) PBMCs, associated with impaired proangiogenic properties that could be rescued by miR-mimic-126 treatment. The present findings provide a novel molecular mechanism underlying increased proangiogenic effects of CD34(+) PBMCs, that is, angiomiR-126 expression/secretion. Moreover, an alteration of angiomiR-126 expression in CD34(+) PBMCs in diabetes provides a novel pathway causing impaired proangiogenic effects.

Tumor-derived galectin-1 (Gal-1), a β-galactoside-binding S-type lectin, has been shown to encourage T-cell death and promote T cell-mediated tumor immune escape. In this report, we show that patients with leukemic cutaneous T-cell lymphomas, known to have limited complexity of their T-cell repertoires, have a predominant T helper type-2 (Th2) cytokine profile and significantly elevated plasma levels of Gal-1 compared with healthy controls. Circulating clonal malignant T cells were a major source of Gal-1. The conditioned supernatant of cultured malignant T cells induced a β-galactoside-dependent inhibition of normal T-cell proliferation and a Th2 skewing of cytokine production. These data implicate Gal-1 in development of the Th2 phenotype in patients with advanced-stage cutaneous T-cell lymphoma and highlight the Gal-1-Gal-1 ligand axis as a potential therapeutic target for enhancing antitumor immune responses.

Signal Transducer and Activator of Transcription 4 (STAT4) is a transcription factor that is activated by IL-12 signaling and promotes Th1-cell differentiation and IFN-γ production. Defective IFN-γ production because of STAT4 mRNA and protein deficiency occurs after autologous stem cell transplantation for lymphoma. In the present study, we investigated the mechanisms of STAT4 deficiency in lymphoma patients. The tumor-bearing state is not responsible, because STAT4 levels were not significantly different in PBMCs obtained from healthy control subjects compared with those from lymphoma patients before treatment. STAT4 protein levels were significantly decreased in PBMCs and T cells obtained from lymphoma patients after standard-dose chemotherapy. Furthermore, treatment of control PBMC cultures or a natural killer cell line with chemotherapy drugs in vitro also resulted in reduced STAT4 protein and diminished, IL-12-induced IFN-γ production. Translation of STAT4 protein was not impaired in chemotherapy-treated cells, whereas the STAT4 protein half-life was significantly reduced. Chemotherapy drugs promoted the ubiquitination and proteasomal degradation of STAT4. Treatment with the proteasome inhibitor bortezomib reversed chemotherapy-induced STAT4 deficiency and defective IFN-γ production. We conclude that acquired STAT4 deficiency in lymphoma patients is a consequence of treatment with chemotherapy, results that have important implications for the design of optimal immunotherapy for lymphoma.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, genetically heterogeneous autosomal recessive immune disorder that results when the critical regulatory pathways that mediate immune defense mechanisms and the natural termination of immune/inflammatory responses are disrupted or overwhelmed. To advance the understanding of FHL, we performed gene expression profiling of peripheral blood mononuclear cells from 11 children with untreated FHL. Total RNA was isolated and gene expression levels were determined using microarray analysis. Comparisons between patients with FHL and normal pediatric controls (n = 30) identified 915 down-regulated and 550 up-regulated genes with more than or equal to 2.5-fold difference in expression (P ≤ .05). The expression of genes associated with natural killer cell functions, innate and adaptive immune responses, proapoptotic proteins, and B- and T-cell differentiation were down-regulated in patients with FHL. Genes associated with the canonical pathways of interleukin-6 (IL-6), IL-10 IL-1, IL-8, TREM1, LXR/RXR activation, and PPAR signaling and genes encoding of antiapoptotic proteins were overexpressed in patients with FHL. This first study of genome-wide expression profiling in children with FHL demonstrates the complexity of gene expression patterns, which underlie the immunobiology of FHL.

Reprogramming blood cells to induced pluripotent stem cells (iPSCs) provides a novel tool for modeling blood diseases in vitro. However, the well-known limitations of current reprogramming technologies include low efficiency, slow kinetics, and transgene integration and residual expression. In the present study, we have demonstrated that iPSCs free of transgene and vector sequences could be generated from human BM and CB mononuclear cells using non-integrating episomal vectors. The reprogramming described here is up to 100 times more efficient, occurs 1-3 weeks faster compared with the reprogramming of fibroblasts, and does not require isolation of progenitors or multiple rounds of transfection. Blood-derived iPSC lines lacked rearrangements of IGH and TCR, indicating that their origin is non-B- or non-T-lymphoid cells. When cocultured on OP9, blood-derived iPSCs could be differentiated back to the blood cells, albeit with lower efficiency compared to fibroblast-derived iPSCs. We also generated transgene-free iPSCs from the BM of a patient with chronic myeloid leukemia (CML). CML iPSCs showed a unique complex chromosomal translocation identified in marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. This approach provides an opportunity to explore banked normal and diseased CB and BM samples without the limitations associated with virus-based methods.

The human T-lymphotropic virus type I (HTLV-I) causes a chronic inflammatory disorder of the central nervous system termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-I encodes a protein known to activate several host-signaling pathways involved in inflammation, such as the nuclear factor-κB (NF-κB). The contribution of the NF-κB pathway to the pathogenesis of HAM/TSP, however, has not been fully defined. We show evidence of canonical NF-κB activation in short-term cultures of peripheral blood mononuclear cells (PBMCs) from subjects with HAM/TSP. NF-κB activation was closely linked to HTLV-I viral protein expression. The NF-κB activation in HAM/TSP PBMCs was reversed by a novel small-molecule inhibitor that demonstrates potent and selective NF-κB antagonist activity. Inhibition of NF-κB activation led to a reduction in the expression of lymphocyte activation markers and resulted in reduced cytokine signaling in HAM/TSP PBMCs. Furthermore, NF-κB inhibition led to a reduction in spontaneous lymphoproliferation, a key ex vivo correlate of the immune activation associated with HAM/TSP. These results indicate that NF-κB activation plays a critical upstream role in the immune activation of HAM/TSP, and identify the NF-κB pathway as a potential target for immunomodulation in HAM/TSP.

Providing humoral immunity, antibody-secreting plasma cells and their immediate precursors, the plasmablasts, are generated in systemic and mucosal immune reactions. Despite their key role in maintaining immunity and immunopathology, little is known about their homeostasis. Here we show that plasmablasts and plasma cells are always detectable in human blood at low frequency in any unimmunized donor. In this steady state, 80% of plasmablasts and plasma cells express immunoglobulin A (IgA). Expression of a functional mucosal chemokine receptor, C-C motif receptor 10 (CCR10) and the adhesion molecule beta(7) integrin suggests that these cells come from mucosal immune reactions and can return to mucosal tissue. These blood-borne, CCR10(+) plasmablasts also are attracted by CXCL12. Approximately 40% of plasma cells in human bone marrow are IgA(+), nonmigratory, and express beta(7) integrin and CCR10, suggesting a substantial contribution of mucosal plasma cells to bone marrow resident, long-lived plasma cells. Six to 8 days after parenteral tetanus/diphtheria vaccination, intracellular IgG(+) cells appear in blood, both CD62L(+), beta(7) integrin(-), dividing, vaccine-specific, migratory plasmablasts and nondividing, nonmigratory, CD62L(-) plasma cells of different specificities. Systemic vaccination does not impact on peripheral IgA(+) plasmablast numbers, indicating that mucosal and systemic humoral immune responses are regulated independent of each other.

CD38 rules proliferation signals in chronic lymphocytic leukemia (CLL) cells, suggesting that the molecule is not merely a prognostic marker but also a key element in the pathogenetic network underlying the disease. CD38 has a genetic polymorphism, characterized by a C>G variation in the regulatory region of intron 1. The working hypothesis is that the presence of different alleles in CLL patients marks (or accounts for) some of the clinical heterogeneity. CD38 allele distribution in 248 Italian patients overlapped with that of the controls (n = 232), suggesting that susceptibility to CLL is not influenced by CD38 genotype. Stratification of patients according to markers of unfavorable prognosis constantly resulted in a significantly higher frequency of the rare G allele. Furthermore, analysis of clinical parameters showed that G allele is independently associated with nodal/splenic involvement. The highest G allele frequency was observed in the 16 patients of the cohort that developed Richter syndrome (RS). Five-year cumulative incidence of transformation was significantly higher in G allele carriers than in CC homozygotes. Multivariate analysis on a total of 30 RS patients confirmed that the probability of transformation is strongly associated with G allele, likely representing an independent risk factor for RS development.

Angiotensin II (Ang-II) exerts inflammatory activity and is involved in different cardiovascular disorders. This study has evaluated the involvement of tumor necrosis factor alpha (TNFalpha) in the leukocyte accumulation elicited by Ang-II. Ang-II (1 nM intraperitoneally in rats) induced TNFalpha release at 1 hour followed by neutrophil and mononuclear cell recruitment. The administration of an antirat TNFalpha antiserum had no effect on Ang-IIinduced neutrophil accumulation but inhibited the infiltration of mononuclear cells and reduced CC chemokine content in the peritoneal exudate. Pretreatment with either an anti-TNFalpha or an anti-IL-4 antiserum decreased Ang-II-induced arteriolar mononuclear leukocyte adhesion by 68% and 60%, respectively, in the rat mesenteric microcirculation. While no expression of TNFalpha was found in the postcapillary venules of Ang-II-injected animals, this cytokine was clearly up-regulated in the arterioles. Stimulation of human umbilical arterial endothelial cells (HUAECs) or isolated human mononuclear cells with 1 microM Ang-II caused increased TNFalpha mRNA expression and protein. Neutralization of TNFalpha activity reduced Ang-II-induced MCP-1, MCP-3, and RANTES release from HUAECs and MIP-1alpha from blood cells. In conclusion, the selective mononuclear leukocyte adhesion to Ang-II-stimulated arterioles is largely mediated by TNFalpha in cooperation with constitutive IL-4. Therefore, neutralization of TNFalpha activity may help to prevent mononuclear cell infiltration and the progression of the atherogenic process.

We examined expression of B cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) and a proliferation-inducing ligand (APRIL) on chronic lymphocytic leukemia (CLL) B cells and nurselike cells (NLCs), which differentiate from CD14+ cells when cultured with CLL B cells. NLCs expressed significantly higher levels of APRIL than monocytes and significantly higher levels of BAFF and APRIL than CLL B cells. Also, the viability of CLL B cells cultured with NLCs was significantly reduced when CLL B cells were cultured with decoy receptor of B-cell maturation antigen (BCMA), which can bind both BAFF and APRIL, but not with BAFF receptor:Fc (BAFF-R:Fc), which binds only to BAFF. The effect(s) of BAFF or APRIL on leukemia cell survival appeared additive and distinct from that of stromal cell-derived factor-1alpha (SDF-1alpha), which in contrast to BAFF or APRIL induced leukemia cell phosphorylation of p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase-1/2 [ERK1/2]) and AKT. Conversely, BAFF and APRIL, but not SDF-1alpha, induced CLL-cell activation of the nuclear factor-kappaB1 (NF-kappaB1) and enhanced CLL-cell expression of the antiapoptotic protein Mcl-1. However, BAFF, but not APRIL, also induced CLL-cell activation of NF-kappaB2. We conclude that BAFF and APRIL from NLCs can function in a paracrine manner to support leukemia cell survival via mechanisms that are distinct from those of SDF-1alpha, indicating that NLCs use multiple distinct pathways to support CLL-cell survival.

Chronic infection or colonization by mycoplasma(s) could gradually and significantly alter many biologic properties of mammalian host cells in culture, including induction of malignant transformation. We examined effects of Mycoplasma fermentans infection on the continuing survival and immortality of human peripheral blood mononuclear cells (PBMCs) from healthy blood donors. Without specific supplemental growth factors, human PBMCs normally die rapidly, with few cells other than macrophages/monocytes surviving after 2 weeks in cultures. Only occasional Epstein-Barr virus (EBV)-positive B lymphocytes would continue to proliferate and undergo spontaneous immortalization. Our present study revealed that infection of human PBMCs in culture with the incognitus and PG18 strains of M fermentans, but surprisingly not with some other strains tested in parallel, markedly enhanced the rate of EBV-positive B lymphocytes to undergo immortalization (74% vs 17%). Compared with spontaneously immortalized PBMCs, the PBMCs immortalized in cultures infected with the mycoplasmas often had prominent karyotype changes with chromosomal loss, gain, or translocations. Furthermore, many of these immortalized B lymphocytes were found to be monoclonal in nature. The in vitro findings would be of relevance to lymphoproliferative disorders that occurred in patients with immune suppression. The mycoplasma-mediated promotional effect in cell immortalization and its potential clinical implications warrant further study.

Common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1; also known as stabilin-1 or FEEL-1) is a large multifunctional glycoprotein implicated in scavenging, angiogenesis, and cell adhesion. Here we studied the function of human CLEVER-1 in leukocyte trafficking. Lymphatic vessels expressed CLEVER-1 constitutively in skin in vivo, whereas on vascular endothelium it appeared only upon inflammation. On isolated vascular endothelial cells, CLEVER-1 supported rolling and transmigration of peripheral blood mononuclear cells (PBMCs) under physiologically relevant laminar shear stress. Intriguingly, CLEVER-1 also mediated transmigration of leukocytes through cultured lymphatic endothelium under static conditions. Thus, synthesis of CLEVER-1 is differentially regulated on the 2 anatomically distinct vascular beds, and CLEVER-1 mediates the transmigration step of the leukocyte traffic in both of them. Notably, CLEVER-1 is the first adhesion molecule shown to be involved in the PBMC transmigration through the lymphatic arm of the immune system.

Type 4 cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE4) inhibitors and other agents that raise intracellular cAMP levels induce apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) but not in T-CLL or peripheral blood T cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and EPAC (exchange protein directly activated by cAMP), a Rap guanosine 5'-diphosphate (GDP) exchange factor. We here examine whether varying expression of EPAC accounts for the discrepant sensitivity of B-CLL and T cells to PDE4 inhibitor-induced apoptosis. B-CLL and peripheral blood B cells express EPAC1 transcript, whereas T-CLL, peripheral blood T cells, monocytes, and neutrophils do not. Treatment with the PDE4 inhibitor rolipram induces Rap1 activation in B-CLL cells but not in peripheral blood B cells, T-CLL, or any of the normal hematopoietic lineages examined. The EPAC-specific cAMP analog 8CPT-2Me-cAMP (8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP) activates Rap1 in B-CLL cells, but, unlike rolipram/forskolin or 8-Bromo-cAMP, it does not induce PKA activation, as judged by phosphorylation of the transcription factor cAMP-response element binding protein (CREB). Unexpectedly, whereas rolipram/forskolin and 8-Bromo-cAMP induce apoptosis in B-CLL cells, 8CPT-2Me-cAMP decreased basal apoptosis in B-CLL cells by an average of 25% (P<.002). Our results demonstrate that B-CLL cells uniquely activate Rap1 in response to PDE4 inhibitors and suggest that physiologic stimuli that activate EPAC may transmit an antiapoptotic signal.

Multiple myeloma (MM) is a progressive disease that is thought to result from multiple genetic insults to the precursor plasma cell that ultimately affords the tumor cell with proliferative potential despite its differentiated phenotype and resistance to undergoing apoptosis. Altered expression of antiapoptotic factors as well as growth factors have been described in a significant number of patients. However, the key regulatory elements that control myeloma development and progression remain largely undefined. Because of the knowledge that B-lymphocyte stimulator (BLyS), a tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis, promotes the survival of malignant B cells, we began a coordinated study of BLyS and its receptors in MM. All MM cells studied expressed one or more of 3 known receptors (B-cell maturation antigen [BCMA], transmembrane activator and CAML interactor [TACI], and B-cell activating factor receptor [BAFF-R]) for BLyS; however, the pattern of expression was variable. Additionally, we provide evidence that BLyS can modulate the proliferative capacity and survival of MM cells. Finally, we provide evidence that BLyS is expressed by MM cells and is present in the bone marrow of patients with MM. Expression of BCMA, TACI, and BAFF-R by MM taken together with the ability of BLyS to support MM cell growth and survival has exciting implications because they may be potential therapeutic targets.

In mammals, it is well documented that observable circadian rhythms are controlled by a central oscillator that is organized in transcriptional and translational feedback loops involving several clock genes. Although recent studies have demonstrated that clock genes oscillate in many peripheral tissues, their characteristics in the human immune system remain unknown. The present study investigates whether circadian clock genes function in human peripheral blood mononuclear cells. On the basis of studies derived from 3 human subjects under controlled conditions, circadian clock genes hPer1, hPer2, hPer3, and hDec1 are expressed in a circadian manner in human peripheral blood mononuclear cells (PBMCs), with the peak level occurring during the habitual time of activity. The demonstration of functional circadian machinery in human PBMCs suggests that peripheral blood cells may be useful for the investigation of human circadian rhythms and their associated disorders.

In a phase 1 dose escalation study, 13 subjects with hemophilia A received by peripheral intravenous infusion a retroviral vector carrying a B-domain-deleted human factor VIII (hFVIII) gene. Infusions were well tolerated. Tests for replication competent retrovirus have been negative. Polymerase chain reaction (PCR) analyses demonstrate the persistence of vector gene sequences in peripheral blood mononuclear cells in 3 of 3 subjects tested. Factor VIII was measured in serial samples using both a one-stage clotting assay and a chromogenic assay. While no subject had sustained FVIII increases, 9 subjects had FVIII higher than 1% on at least 2 occasions 5 or more days after infusion of exogenous FVIII, with isolated levels that ranged from 2.3% to 19%. Pharmacokinetic parameters of exogenous FVIII infused into subjects 13 weeks after vector infusion showed an increased half-life (T1/2; P <.02) and area under the curve (AUC, P <.04) compared with prestudy values. Bleeding frequency decreased in 5 subjects compared with historical rates. These results demonstrate that this retroviral vector (hFVIII(V)) is safe and, in some subjects, persists more than a year in peripheral blood mononuclear cells, with measurable factor VIII levels and with increased available FVIII activity (increased T1/2 and AUC) after infusion of exogenous FVIII concentrate.

Migration of antigen (Ag)-loaded dendritic cells (DCs) from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. We evaluated monocyte-derived DCs (MoDCs) and peripheral blood DCs (PBDCs) to respond to proinflammatory mediators, CD40L, and intact bacteria. All classes of stimuli induced DC phenotypic maturation. However, for MoDCs, only prostaglandin E(2) (PGE(2))-containing stimuli induced migratory-type DCs. Thus, immature MoDCs that encountered proinflammatory cytokines or CD40L or intact bacteria in the presence of PGE(2) acquired migratory capacity but secreted low levels of cytokines. Conversely, MoDCs that encountered pathogens or CD40L alone become nonmigratory cytokine-secreting cells (proinflammatory type). Interestingly, both migratory- and proinflammatory-type DCs expressed equivalent levels of chemokine receptors, suggesting that the role of PGE(2) was to switch on migratory function. We demonstrate that PGE(2) induces migration via the E-prostanoid 2/E-prostanoid 4 (EP(2)/EP(4)) receptors and the cAMP pathway. Finally, migratory-type MoDCs stimulated T-cell proliferation and predominantly IL-2 secretion, whereas proinflammatory-type MoDCs induced IFN-gamma production. In contrast, CD1b/c(+) PBDC rapidly acquired migratory capacity irrespective of the class of stimulus encountered and secreted low levels of cytokines. This suggests that not all mature stages of DCs are destined to migrate to lymphoid organs and that the sequence in which stimuli are encountered significantly affects which functions are expressed. Thus, certain immature DC subsets recruited from the resting precursor pool may have multiple functional fates that play distinct roles during the induction and effector phases of the immune response. These findings have important implications for the clinical utility of DCs in immunotherapy.

Sickle cell anemia is characterized by painful vaso-occlusive crises. It is hypothesized that monocytes are activated in sickle cell disease and can enhance vaso-occlusion by activating endothelium. To test this hypothesis, human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (MVEC) with sickle and normal mononuclear leukocytes were incubated, and endothelial activation was measured. Endothelial cells incubated with sickle mononuclear leukocytes were more activated than those incubated with normal mononuclear leukocytes, as judged by the increased endothelial expression of adhesion molecules and tissue factor and the adhesion of polymorphonuclear leukocytes (PMNL). Monocytes, not lymphocytes or platelets, were the mononuclear cells responsible for activating endothelial cells. Sickle monocytes triggered endothelial nuclear factor-kappa B (NF-kappaB) nuclear translocation. Cell-to-cell contact of monocytes and endothelium enhanced, but was not required for, activation. Antibodies to tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) blocked activation of the endothelium by monocytes. Peripheral blood monocytes from patients with sickle cell disease had 34% more IL-1beta (P =.002) and 139% more TNF-alpha (P =.002) per cell than normal monocytes. Sixty percent of sickle monocytes expressed the adhesion molecule ligand CD11b on their surfaces compared with only 20% of normal monocytes (P =.002). Serum C-reactive protein, a marker of systemic inflammation, was increased 12-fold in sickle serum than in normal serum (P =.003). These results demonstrate that sickle monocytes are activated and can, in turn, activate endothelial cells. It is speculated that vascular inflammation, marked by activated monocytes and endothelium, plays a significant role in the pathophysiology of vaso-occlusion in sickle cell anemia.

CD40 antigen is a costimulatory molecule highly expressed on dendritic cells (DC) and activated B cells, which induces T-cell proliferation through the binding with CD40L receptor. In this study, we evaluated CD40 expression on normal CD34(+) blood cells and functionally characterized CD34(+)CD40(+) and CD34(+)CD40(-) cell subsets. CD40, CD80, and CD86 antigens were constitutively expressed on 3.2% +/- 4.5%, 0%, and 1.8% +/- 1.2% CD34(+) blood cells, respectively. However, after 24 hours in liquid culture with medium alone, or with tumor-necrosis-factor-alpha (TNF-alpha), or with allogeneic mononuclear cells 10.8% +/- 3.8%, 75.3% +/- 15.0% and 53. 7% +/- 17.0% CD34(+) blood cells, respectively, became CD40(+). After incubation for 24 hours with TNF-alpha CD34(+)CD40(+) blood cells expressed only myeloid markers and contained less than 5% CD86(+) and CD80(+) cells. Also, a 24-hour priming with TNF-alpha or ligation of CD40 significantly increased the CD34(+) blood cells alloantigen presenting function. Finally, purified CD34(+)CD40(+) blood cells stimulated an alloreactive T-cell response in MLC, were enriched in granulocytic, monocytic, and dendritic precursors, and generated high numbers of DC in 11-14 d liquid cultures with GM-CSF, SCF, TNF-alpha and FLT-3L. In contrast, CD34(+)CD40(-) cells were poorly immunogenic, contained committed granulocytic and erythroid precursors and early progenitors, and differentiated poorly toward the DC lineage. In conclusion, a short incubation with TNF-alpha allows the selection of CD40(+) blood progenitors, which may be a useful source of DC precursors for antitumor vaccine studies, and also a CD34(+)CD40(-) blood cell fraction that could be exploited in innovative strategies of allogeneic transplantation across HLA barriers.

CD95 (Fas)-induced apoptosis plays a critical role in the elimination of activated lymphocytes and induction of peripheral tolerance. Defects in CD95/CD95L (Fas-Ligand)-apoptotic pathway have been recognized in autoimmune lymphoproliferative diseases (ALPS) and lpr or gld mice and attributed to CD95 and CD95L gene mutations, respectively. Large granular lymphocyte (LGL) leukemia is a chronic disease characterized by a proliferation of antigen-activated cytotoxic T lymphocytes. Autoimmune features such as hypergammaglobulinemia, rheumatoid factor, and circulating immune complexes are common features in LGL leukemia and ALPS. Therefore, we hypothesize that expansion of leukemic LGL may be secondary to a defective CD95 apoptotic pathway. In this study, we investigated expression of CD95 and CD95L in 11 patients with CD3(+) LGL leukemia and explored the apoptotic response to agonistic CD95 monoclonal antibody (MoAb). We found that leukemic LGL from each patient expressed constitutively high levels of CD95/CD95L, similar to those seen in normal activated T cells. However, cells from 9 of these 11 patients were totally resistant to anti-CD95-induced apoptosis. Similarly, cells were resistant to anti-CD3-MoAb-triggered cell death. Lack of anti-CD95-induced apoptosis was not due to mutations in the CD95 antigen. Leukemic LGL were not intrinsically resistant to CD95-dependent death, because LGL from all but 1 patient underwent apoptosis after phytohemagglutinin/interleukin-2 activation. The patient whose leukemic LGL were intrinsically resistant to CD95 had an aggressive form of LGL leukemia that was resistant to combination chemotherapy. These findings that leukemic LGL are resistant to CD95-dependent apoptosis despite expressing high levels of CD95 are similar to observations made in CD95L transgenic mice. These data suggest that LGL leukemia may be a useful model of dysregulated apoptosis causing human malignancy and autoimmune disease.

Human umbilical vein endothelial cells (HUVECs) undergo programmed cell death (apoptosis) after coculture with peripheral blood mononuclear cells (PBMCs) preactivated by ionizing radiation (IR) or by bacterial endotoxin (lipopolysaccharide [LPS]). Cell-to-cell contact-mediated apoptosis could be blocked in both cases by anti-tumor necrosis factor-alpha (anti-TNF-alpha) monoclonal antibody MAK195 and also by the antagonistic cytokine interleukin-10 (IL-10). Cell-free PBMC supernatants from both preactivation treatments were sufficient to trigger endothelial apoptosis. In contrast, MAK195 and IL-10 were found to be ineffective in this system, suggesting a TNF-alpha-independent mechanism. However, N-Acetylcystein, an antioxidant, fully abrogated programmed cell death mediated by the supernatant of IR-treated PBMCs, but not of LPS-treated PBMCs. Additionally, we found that coculture and cell-free supernatants of preactivated as well as untreated PBMCs caused cell cycle arrest in proliferating EC in G(0/1), which could be relieved by IL-10, but not by anti-TNF-alpha. Further analysis showed that transforming growth factor-beta, which was constitutively expressed in the supernatant of PBMCs, namely lymphocytes, was responsible for this. These data suggest a pathophysiologic model in which preactivated PBMCs cause EC damage and may prevent blood vessel repair by arresting the proliferation of ECs. This could contribute to the understanding of various clinical endothelial complications that occur after irradiation as well as in cases of endotoxemia or related inflammatory states.

Natural killer cells (NK) were studied to determine factors important in their expansion. Flourescence-activated cell sorter (FACS) purified CD56+/CD3- NK cells cultured alone for 18 days in rIL-2 containing medium (1,000 U/mL) showed enhanced cytotoxicity but only minimal expansion. NK expansion was increased (12.5 +/- 1.6-fold) by coculturing NK with soluble factors produced by irradiated peripheral blood mononuclear cells (PBMNC) in which the two populations were separated by a microporous membrane. However, maximal NK expansion was always observed when NK were cocultured in direct contact with irradiated PBMNC (49.4 +/- 5.9-fold). To determine if marrow stroma, which supports differentiation of primitive NK progenitors, was a better accessory cell population than irradiated PBMNC, NK were cocultured in direct contact with primary marrow stromal layers. NK expansion with marrow stroma was similar to PBMNC. Fibroblast cell lines (M2-10B4, NRK-49F, NIH-3T3) and human umbilical vein endothelial cells (HUVEC), all homogeneous populations and devoid of monocytes, also exhibited a similar contact-dependent increase in NK expansion. Experiments were designed using fixed M2-10B4 stromal cells to separate the contact-induced proliferative stimuli from soluble factors. NK plated directly on ethanol/acetic acid-fixed M2-10B4, which leaves stromal ligands (cell membrane components and ECM) intact, resulted in increased NK expansion compared with medium alone. We further show that the combination of independent contact and soluble factors is responsible for maximal late NK expansion (days 28 through 40) but paradoxically inhibits early NK expansion (day 7). The proliferation inhibitory effects were verified by 3H-thymidine uptake and could be detected at days 2 through 6 but no longer 14 days after the initiation of the culture. We show that both laminin and thrombospondin inhibit early NK proliferation, whereas only thrombospondin was capable of also stimulating late NK expansion. The effect of thrombospondin on early NK proliferation is related to activation of transforming growth factor-beta 1 (TGF-beta) because anti-TGF-beta neutralizing antibody completely abrogated thrombospondin-mediated inhibition of early NK proliferation. Although inhibitory early in culture, active TGF-beta added only at culture initiation increases late NK expansion similar to thrombospondin. TGF-beta was not present in the thrombospondin preparation but latent TGF-beta in serum, or TGF-beta transcripts identified in IL-2-activated NK could explain paracrine or autocrine mechanisms for the regulation of NK proliferation. Finally, anti-TGF-beta neutralizing antibody only minimally affects stroma-mediated inhibition of early NK proliferation suggesting that aside from thrombospondin/TGF-beta, additional contact factors are important for the regulation of NK proliferation.

Interactions of hematopoietic progenitor cells (HPC) with bone marrow stroma, mediated by adhesion molecules, are assumed to be critically important to the regulation of hematopoiesis. However, the specific roles of individual adhesion molecules involved in these interactions are poorly understood. Here, a monoclonal antibody, MEM-59, recognizing CD43, an adhesion molecule highly expressed on HPC, is shown to induce apoptosis in this cell population. This process operates at the single-cell level, and its initiation requires crosslinking of surface CD43 and the presence of cytokines. In contrast to HPC, more differentiated cells originating from this primitive cell population, as well as peripheral lymphocytes, do not undergo apoptosis in response to the CD43-mediated stimulation. Thus, CD43 may function as a negative regulator of early hematopoietic events, delivering a signal for apoptosis of HPC.

This study investigates apoptosis as a mechanism for CD4+ T-cell depletion in human immunodeficiency virus type-1 (HIV-1) infection. Although several recent studies have shown that T cells of HIV-infected individuals show enhanced susceptibility to cell death by apoptosis, the mechanisms responsible for apoptosis are largely unknown. By using a flow cytometric technique and by morphology, we have quantitated the percentage of cells undergoing apoptosis in peripheral blood mononuclear cells (PBMCs) from HIV-seronegative donors and from HIV-infected asymptomatic patients. The PBMCs were cultured without any stimulus or with staphylococcus enterotoxin B, anti-T-cell receptor (TCR) alpha beta monoclonal antibody WT-31, or phytohemagglutinin for periods up to 6 days. In addition, we sought to determine whether cross-linking of CD4 followed by various modes of TCR stimulation in vitro could induce apoptosis in normal PBMCs. Here we show that (1) patient PMBCs undergo marked spontaneous apoptosis; (2) stimulation of T cells of patients as well as normal donors results in increased apoptosis; and (3) cross-linking of CD4 molecules is sufficient to induce apoptosis in CD4+ T cells if cross-linking is performed in unfractioned PBMCs, but not if CD4 molecules are cross-linked in purified T-cell preparations. These observations strongly suggest that accelerated cell death through apoptosis plays an important role in the pathogenesis of HIV-1 infection. At the same time, our observations implicate cross-linking of CD4 in vivo as a major contributor to this mechanism of accelerated cell death in HIV infection.

Various immunologic stimuli and heterologous viral regulatory elements have been shown to increase susceptibility to, and replication of, human immunodeficiency virus type 1 (HIV-1) in lymphocytes and monocytes in vitro. Transfusion of allogeneic blood components from heterologous donors constitutes a profound immunologic stimulus to the recipient, in addition to being a potential route of transmission of lymphotropic viral infections. To investigate the hypothesis that transfusions, and particularly those containing leukocytes, activate HIV-1 replication in infected recipient cells, we cocultured peripheral blood mononuclear cells (PBMC) from three anti-HIV-1-positive individuals with allogeneic donor PBMC, as well as partially purified populations of donor lymphocytes, monocytes, granulocytes, platelets, and red blood cells (RBC) and allogeneic cell-free plasma. Allogeneic PBMC induced a dose-related activation of HIV-1 expression in in vivo infected cells, followed by dissemination of HIV-1 to previously uninfected patient cells. Activation of HIV-1 replication was observed with donor lymphocytes, monocytes, and granulocytes, whereas no effect was seen with leukocyte-depleted RBC, platelets, or plasma (ie, therapeutic blood constituents). Allogeneic donor PBMC were also shown to upregulate HIV-1 expression in a "latently" infected cell line, and to increase susceptibility of heterologous donor PBMC to acute HIV-1 infection. Studies should be performed to evaluate whether transfusions of leukocyte-containing blood components accelerate HIV-1 dissemination and disease progression in vivo. If so, HIV-1-infected patients should be transfused as infrequently as possible and leukocyte-depleted (filtered) blood components should be used to avoid this complication.

The NADPH:O2 oxidoreductase of phagocytic leukocytes is an important enzyme for the bactericidal activity of these cells. Cytochrome b558 is a membrane component of this enzyme. In X-linked chronic granulomatous disease (Xb- CGD) the phagocytes are defective in the beta-subunit (gp91-phox) of this cytochrome. We have studied the genetic defect in a group of six X-linked CGD patients characterized by complete or partial loss of cytochrome b558 with the use of the polymerase chain reaction. All patients had a different single point mutation in the gp91-phox gene, indicating that the genetic defect in Xb- CGD is very heterogeneous. In one patient the mutation leads to a premature termination codon. In the other five cases these mutations predict incorporation of a different amino acid. The mutations were with one exception found in the N-terminal half of the protein, suggesting that this part of cytochrome b558 is important for the binding of the heme or for formation of a stable complex with p22-phox. Two histidyl residues were found that might be ligands of the heme iron.

Pentoxifylline (Trental), used routinely for the treatment of intermittent claudication, has been shown previously to decrease the levels of tumor necrosis factors-alpha (TNF-alpha) RNA in cancer patients and to lead to a general improvement of well being. Increased TNF-alpha levels have been observed not only in cancer patients but also in cachectic patients with the acquired immunodeficiency syndrome (AIDS), and TNF-alpha is known to increase the expression of the human immunodeficiency virus type 1 (HIV-1) via activating its long terminal repeat (LTR). Moreover, TNF-alpha decreases the therapeutic efficacy of zidovudine (AZT). Here we show a significant decrease in HIV-1 replication by pentoxifylline in infected human peripheral blood mononuclear cells. The reduction was proportional to the downregulation of expression of a reporter gene, the bacterial gene for chloramphenicol acetyl transferase, linked to the HIV-1 LTR in human monocytoid cells. We conclude that patients with AIDS may benefit from pentoxifylline treatment because of its blockage of TNF-alpha-mediated HIV-1 upregulation, from increased efficacy of AZT, and also from improvement in TNF-alpha-induced cachexia.

High concentrations of tumor necrosis factor (TNF) alpha have been detected in the plasma of patients undergoing immunotherapy with interleukin 2 (IL-2), suggesting that this cytokine may play a role in the fever and shocklike state induced by the administration of high-dose IL-2. Dexamethasone has been shown to inhibit the synthesis of TNF by monocytes activated in vitro by endotoxin. To determine if dexamethasone can exert a similar suppressive effect on IL-2-induced TNF synthesis in vivo, the concentration of TNF alpha was measured in plasma samples serially obtained (a) from cancer patients participating in a phase I dose escalation clinical trial with high-dose IL-2 administered in conjunction with dexamethasone (IL-2/Dex) and (b) from patients participating in concurrent studies with IL-2 alone. In contrast to the high plasma levels of TNF alpha detected in patients receiving IL-2 alone, TNF levels in most of the IL-2/Dex patients remained below the threshold of detectability of our TNF radioimmunoassay. The concurrent administration of dexamethasone also prevented the IL-2-induced increase in serum levels of C-reactive protein, a hepatic acute phase reactant whose synthesis is regulated by proinflammatory cytokines such as TNF. The steroid-treated patients also failed to develop the neutrophil chemotactic defect characteristic of IL-2 recipients. The concomitant administration of dexamethasone increased the maximum tolerated dose of IL-2 approximately threefold and markedly reduced the hypotension and organ dysfunction ordinarily observed in these patients. These results demonstrate that dexamethasone inhibits the release of TNF into the circulation of patients undergoing immunotherapy with IL-2. They further suggest that the altered spectrum and reduced severity of IL-2 side effects observed in patients receiving dexamethasone may be attributable in part to the suppressive effect of steroids on IL-2-induced TNF synthesis.

We studied the in vitro effects of lymphokine-activated killer (LAK) cells from the peripheral blood of chronic myeloid leukemia (CML) patients after allogeneic and syngeneic bone marrow transplantation (BMT). LAK cells were generated by incubating peripheral blood mononuclear cells from patients post-BMT with recombinant interleukin-2 (IL-2) (500 U/mL) in 10% AB serum for 7 days. They were phenotyped and tested for activity in a standard 4-hour 51Cr release assay (n = 37) and in a CFU-GM assay (n = 24). We found that the LAK cells were mainly activated natural killer cells, but some were CD3+ T cells. In the 51Cr release assay LAK cells from 20 of 33 (61%) allogeneic and 2 of 4 syngeneic recipients killed recipient CML cells and in 22 of 37 (60%) cases also killed the HLA disparate CML cells. In the CFU-GM assay the LAK cells incubated together with the CML cells in liquid culture before plating inhibited (P less than .05) colony growth in 16 of 22 allogeneic and 2 of 2 syngeneic recipients. Cell-cell contact was necessary for optimal effect. There was little or no inhibition of proliferation of donor marrow CFU-GM. This in vitro graft-versus-leukemia (GVL) effect could also be demonstrated after LAK effectors were depleted of CD3+ T cells. It was inducible in recipients of both T cell-depleted and T cell-replete donor marrow and in recipients with or without graft-versus-host disease. These results suggest that a major histocompatibility complex-unrestricted GVL effect is inducible following allogeneic and syngeneic BMT. The use of IL-2/LAK cells after BMT could reduce the risk of relapse.

Patients who undergo allogeneic bone marrow transplantation (BMT) are clinically immunodeficient for a prolonged period after engraftment. In the present study, we examined immune function after BMT in a series of patients who had received HLA compatible sibling marrow grafts purged of T cells with anti-CD6 monoclonal antibody and complement. None of the patients in this analysis received immunomodulating agents and none had developed graft-versus-host disease (GVHD). Initially after BMT, natural killer (NK) cells are the predominant cell type, giving way to CD3+, CD5+ T cells after 4 to 8 weeks. Despite the return of normal numbers of T lymphocytes post-BMT phenotypic analysis reveals several long-term abnormalities, including an inverted T4:T8 ratio and a significant fraction of CD3+ T cells that do not co-express CD6. In mitogenic assays, stimulation by either nonspecific lectin (phytohemagglutinin; PHA) or antibodies to the CD2 surface structure (anti-T11(2) + anti-T11(3)) results in decreased levels of T-cell proliferation compared with controls for over 18 months post-BMT. In contrast, the ability of unstimulated peripheral blood mononuclear cells (PBMC) to respond to recombinant interleukin-2 (rIL-2) is relatively intact, most likely reflecting early functional reconstitution of the NK cell population. To further characterize the prolonged abnormalities in T-cell proliferation after PHA or CD2 stimulation, we examined more proximal events in T-cell activation such as induction of IL-2 receptor expression and stimulus-induced intracellular calcium flux. We found that the induction of IL-2 receptor (p55) after in vitro activation, although initially abnormal, recovers completely by 6 months post-BMT. We also found that, after CD2 stimulation, calcium flux in T cells was normal immediately after engraftment. In contrast, after stimulation with anti-CD3 antibodies, a large population of T cells do not develop intracellular calcium flux compared with controls. We conclude that despite the recovery of normal numbers of T lymphocytes early after engraftment of CD6-depleted marrow, these T cells exhibit several physiologic and functional abnormalities that persist for varying intervals post-BMT. At present, it is unclear which of these specific defects is most closely associated with increased susceptibility to infectious agents after BMT.

The receptors for fibronectin (FN-R) and vitronectin (VN-R) belong to a family of integral membrane glycoproteins known to be involved in cell-extracellular matrix and cell-cell interactions named integrins (FN-R = beta 1 integrin and VN-R = beta 3 integrin). Adhesion studies using FN-coated plastic dishes and highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) showed a strong binding of monocytes and T lymphocytes to FN but virtually no binding of B cells to FN. Binding of monocytes and T cells to FN could be partially inhibited by a hexapeptide (GRGDSP) containing the adhesive peptide sequence Arg-Gly-Asp (RGD) as well as by an anti-FN-R antibody. The distribution of beta 1 and beta 3 integrin complexes on PBMCs was characterized by immunoprecipitation of detergent extracts of 125I-labeled cells using polyclonal antibodies against these two receptors. Two surface polypeptides corresponding to the alpha and beta chains of FN-R and VN-R were found on all three cell types. To characterize these receptors further, monoclonal antibodies (MoAbs) against the very late antigens (VLAs) 1, 3, and 5 were used for immunoprecipitation studies. Monocytes and T cells reacted with VLA 5 that was previously identified as the human FN receptor, whereas no labeling with anti-VLA 5 could be shown for B cells. When cell populations were cultured in 10% human serum for 24 hours, an increase in beta 1-integrin+ monocytes and T cells was observed. The number of beta 3-integrin+ cells remained essentially unchanged. The presence of beta 1 and beta 3 integrins on monocytes as well as on T and B lymphocytes may be of significance in the ability of these cells to interact with each other and participate in hematopoiesis and certain immune reactions.

The feasibility of in vitro interleukin 2 (IL-2) activation and expansion of mononuclear cells (MNCs) derived from adult patients with acute myelogenous leukemia (ANLL) was studied. Patients' natural killer (NK) and lymphokine-activated killer (LAK) cell activity was compared with that of normal donors in terms of: (a) cytolytic activity (four-hour 51Cr release assay) against an NK-sensitive target (K562), NK-resistant targets (Raji/Daudi), and fresh/cryopreserved autologous and allogeneic leukemic blasts; (b) proliferation and expansion in culture with 1,000 U/mL recombinant IL 2 (rIL 2); and (c) the cell surface phenotype of the cultured cells. In 21 of 24 patients with active disease (AP) MNCs derived from the peripheral blood (PBL) or bone marrow (BM) could be cultured and expanded in the presence of rIL 2. These cultures initially contained between 30% and 50% blasts, and during 2 to 4 weeks of culture destruction of blasts and enrichment of up to 60% in cells with the morphology of large granular lymphocytes (LGLs) was observed. Expansion in culture varied between two- and 100-fold. MNCs from all patients in remission (RP) could be activated by rIL 2 and expanded up to 30-fold after 1 to 3 weeks in culture. NK activity of fresh PBLs from AP was significantly lower than in normal controls, whereas NK activity of RP was within the normal range. High levels of postactivation NK and LAK activity on K562/Raji/Daudi and on fresh/cryopreserved leukemic blasts was generated in approximately 50% of cases of AP and in most RP. Cell surface phenotype studies showed that cultured cells derived from ANLL patients were significantly enriched (up to 40%) in NKH-1 (Leu 19) positive cells, with RP LAK cells also expressing a high proportion of CD16 positive cells (up to 40%). This study has shown that it is feasible to activate and significantly expand killer cells derived from active disease and remission ANLL patients during 1 to 3 weeks culture with IL 2 with good maintenance of cytolytic activity. Both initial NK activity and LAK generation was optimal in remission patients. Based on data from this study, a clinical protocol has been developed for treatment of early relapse ANLL patients with LAK cells cultured for 1 to 3 weeks and systemic IL 2.

Human peripheral blood mononuclear cells (PBM) respond to lipopolysaccharide (LPS) with increased release of a plasminogen activator (PA) inhibitor. This response is dose dependent and parallels the LPS-induced expression of PBM tissue factor activity. The PA inhibitors of control and LPS-stimulated PBMs appear identical as both are identified by antibodies to PA inhibitor type 2 of human placenta, but not by antibodies to type 1 inhibitor of bovine aortic endothelial cells. The PA inhibitor is specific for urokinase type PA as determined by the 125I-fibrin plate assay, and direct cleavage of 125I-plasminogen; it does not effectively inhibit tissue-type PA. The inhibitor forms an active site-dependent complex with 125I-urokinase, which then demonstrates an increase in mol wt from 33 kd to 68 kd on reduced sodium dodecyl sulfate (SDS) polyacrylamide gels. PBMs neither secrete nor express active PA. Hence, the exposure of PBMs to LPS results in conditions highly favorable to fibrin deposition and persistence: increased procoagulant and antifibrinolytic activities, accompanied by no measurable PA. Such modulation of these effectors may be important in the pathogenesis of fibrin characteristically found in tissue lesions of endotoxin-initiated processes.