Therapeutic plasma exchange for neonatal hepatic failure.

We report a case of therapeutic plasma exchange in a neonate with fulminant liver failure. A six-day old, 2800-gram baby was referred to our medical center for evaluation and treatment of fulminant hepatic failure. The working diagnosis at admission was gestational alloimmune liver disease, and therapeutic plasma exchange was proposed. A double volume plasma exchange was successfully performed, using the Spectra Optia apheresis system, primed with packed red blood cells. Access was obtained via a radial artery catheter and a peripheral intravenous line. On hospital D-14 a diagnosis of E3 deficiency was confirmed, and disease-specific therapy was started. Automated TPE using peripheral arterial and venous catheters may be safely performed in neonates, and should be considered in the treatment of a variety of disorders including neonatal fulminant hepatic failure.

Tumor necrosis factor receptors support murine hematopoietic progenitor function in the early stages of engraftment.

Tumor necrosis factor (TNF) family receptors/ligands are important participants in hematopoietic homeostasis, in particular as essential negative expansion regulators of differentiated clones. As a prominent injury cytokine, TNF-alpha has been traditionally considered to suppress donor hematopoietic stem and progenitor cell function after transplantation. We monitored the involvement of TNF receptors (TNF-R) 1 and 2 in murine hematopoietic cell engraftment and their inter-relationship with Fas. Transplantation of lineage-negative (lin(-)) bone marrow cells (BMC) from TNF receptor-deficient mice into wild-type recipients showed defective early engraftment and loss of durable hematopoietic contribution upon recovery of host hematopoiesis. Consistently, cells deficient in TNF receptors had reduced competitive capacity as compared to wild-type progenitors. The TNF receptors were acutely upregulated in bone marrow (BM)-homed donor cells (wild-type) early after transplantation, being expressed in 60%-75% of the donor cells after 6 days. Both TNF receptors were detected in fast cycling, early differentiating progenitors, and were ubiquitously expressed in the most primitive progenitors with long-term reconstituting potential (lin(-)c-kit(+) stem cell antigen (SCA)-1(+)). BM-homed donor cells were insensitive to apoptosis induced by TNF-alpha and Fas-ligand and their combination, despite reciprocal inductive cross talk between the TNF and Fas receptors. The engraftment supporting effect of TNF-alpha is attributed to stimulation of progenitors through TNF-R1, which involves activation of the caspase cascade. This stimulatory effect was not observed for TNF-R2, and this receptor did not assume redundant stimulatory function in TNFR1-deficient cells. It is concluded that TNF-alpha plays a tropic role early after transplantation, which is essential to successful progenitor engraftment.

Expression of Fas and Fas-ligand in donor hematopoietic stem and progenitor cells is dissociated from the sensitivity to apoptosis.

OBJECTIVE: The interaction between the Fas receptor and its cognate ligand (FasL) has been implicated in the mutual suppression of donor and host hematopoietic cells after transplantation. Following the observation of deficient early engraftment of Fas and FasL-defective donor cells and recipients, we determined the role of the Fas-FasL interaction. METHODS: Donor cells were recovered after syngeneic (CD45.1-->CD45.2) transplants from various organs and assessed for expression of Fas/FasL in reference to lineage markers, carboxyfluorescein succinimidyl ester dilution, Sca-1 and c-kit expression. Naïve and bone marrow-homed cells were challenged for apoptosis ex vivo. RESULTS: The Fas receptor and ligand were markedly upregulated to 40% to 60% (p < 0.001 vs 5-10% in naïve cells) within 2 days after syngeneic transplantation, while residual host cells displayed modest and delayed upregulation of these molecules ( approximately 10%). All lin(-)Sca(+)c-kit(+) cells were Fas(+)FasL(+), including 95% of Sca-1(+) and 30% of c-kit(+) cells. Fas and FasL expression varied in donor cells that homed to bone marrow, spleen, liver and lung, and was induced by interaction with the stroma, irradiation, cell cycling, and differentiation. Bone marrow-homed donor cells challenged with supralethal doses of FasL were insensitive to apoptosis (3.2% +/- 1% vs 38% +/- 5% in naïve bone marrow cells), and engraftment was not affected by pretransplantation exposure of donor cells to an apoptotic challenge with FasL. CONCLUSION: There was no evidence of Fas-mediated suppression of donor and host cell activity after transplantation. Resistance to Fas-mediated apoptosis evolves as a functional characteristic of hematopoietic reconstituting stem and progenitor cells, providing them competitive engraftment advantage over committed progenitors.

Fas transduces dual apoptotic and trophic signals in hematopoietic progenitors.

Stem cells and progenitors are often required to realize their differentiation potential in hostile microenvironments. The Fas/Fas ligand (FasL) interaction is a major effector pathway of apoptosis, which negatively regulates the expansion of differentiated hematopoietic cells. The involvement of this molecular interaction in the function of hematopoietic stem and progenitor cells is not well understood. In the murine syngeneic transplant setting, both Fas and FasL are acutely upregulated in bone marrow-homed donor cells; however, the Fas(+) cells are largely insensitive to FasL-induced apoptosis. In heterogeneous populations of lineage-negative (lin(-)) bone marrow cells and progenitors isolated by counterflow centrifugal elutriation, trimerization of the Fas receptor enhanced the clonogenic activity. Inhibition of caspases 3 and 8 did not affect the trophic signals mediated by Fas, yet it efficiently blocked the apoptotic pathways. Fas-mediated tropism appears to be of physiological significance, as pre-exposure of donor cells to FasL improved the radioprotective qualities of hematopoietic progenitors, resulting in superior survival of myeloablated hosts. Under these conditions, the activity of long-term reconstituting cells was not affected, as determined in sequential secondary and tertiary transplants. Dual caspase-independent tropic and caspase-dependent apoptotic signaling place the Fas receptor at an important junction of activation and death. This regulatory mechanism of hematopoietic homeostasis activates progenitors to promote the recovery from aplasia and converts into a negative regulator in distal stages of cell differentiation. Disclosure of potential conflicts of interest is found at the end of this article.

Pancreatic islets under attack: cellular and molecular effectors.

Abundant information is available on the involvement of various cellular and molecular mechanisms in beta cell apoptosis. The experimental evidence is controversial and difficult to reconcile, and the mechanisms of evasion of the autoreactive clones from immune surveillance are poorly understood. Multiple apoptotic pathways play a role in destructive insulitis, including perforin/granzyme, Fas/Fas-ligand (FasL), and other members of the necrosis factor superfamily. These pathways present redundant behaviors in both the initial and late stages of beta cell injury, and at the same time, each molecular mechanism is dispensable in the evolution of autoimmune diabetes. There may be a preferential use of perforin/granzyme in CD8(+) T cell-mediated lysis, which participates in onset of autoimmunity, and a predominance of FasL in CD4(+) T cell-mediated insulitis. Several cytokines released in the inflammatory infiltrate induce Fas expression in beta cells, priming them to FasL-mediated apoptosis. In this review, we focus on the possible participation of multiple cell subsets and molecular mechanisms in the pathogenesis of diabetes to the point where inflammation incites an irreversible vicious cycle that perpetuates beta cell death.

Fas ligand enhances hematopoietic cell engraftment through abrogation of alloimmune responses and nonimmunogenic interactions.

Early after transplantation, donor lineage-negative bone marrow cells (lin(-) BMC) constitutively upregulated their expression of Fas ligand (FasL), suggesting an involvement of the Fas/FasL axis in engraftment. Following the observation of impaired engraftment in the presence of a dysfunctional Fas/FasL axis in FasL-defective (gld) donors or Fas-defective (lpr) recipients, we expressed a noncleavable FasL chimeric protein on the surface of donor lin(-) BMC. Despite a short life span of the protein in vivo, expression of FasL on the surface of all the donor lin(-) BMC improved the efficiency of engraftment twofold. The FasL-coated donor cells efficiently blunted the host alloimmune responses in primary recipients and retained their hematopoietic reconstituting potential in secondary transplants. Surprisingly, FasL protein improved the efficiency of engraftment in syngeneic transplants. The deficient engraftment in lpr recipients was not reversed in chimeric mice with Fas(-) stroma and Fas(+) BMC, demonstrating that the host marrow stroma was also a target of donor cell FasL. Hematopoietic stem and progenitor cells are insensitive to Fas-mediated apoptosis and thus can exploit the constitutive expression of FasL to exert potent veto activities in the early stages of engraftment. Manipulation of the donor cells using ectopic FasL protein accentuated the immunogenic and nonimmunogenic interactions between the donor cells and the host, alleviating the requirement for a megadose of transplanted cells to achieve a potent veto effect. Disclosure of potential conflicts of interest is found at the end of this article.

Interferon-gamma and bacterial lipopolysaccharide act synergistically on human neutrophils enhancing interleukin-8, interleukin-1beta, tumor necrosis factor-alpha, and interleukin-12 p70 secretion and phagocytosis via upregulation of toll-like receptor 4.

In human neutrophils, interferon (IFN)-gamma enhanced the expression of toll-like receptor 4 (TLR4), a crucial component of the signaling receptor complex for bacterial lipopolysaccharide (LPS). Lipopolysaccharide alone did not affect TLR4 expression, but costimulation with IFN-gamma and LPS induced higher levels of TLR4 expression than stimulation with IFN-gamma alone. Using the protein synthesis inhibitor cycloheximide and measuring the expression of CD35 in neutrophils stimulated with IFN-gamma and LPS alone or in combination, we could demonstrate that IFN-gamma enhances TLR4 by de novo protein synthesis, whereas the addition of LPS acts synergistically by enhancing vesicular mobilization to the cell surface. Costimulation with IFN-gamma and LPS induced neutrophil activation and enhanced secretion of the cytokines, interleukin (IL)-8, IL-1beta, tumor necrosis factor-alpha, and IL-12 p70, and phagocytosis of latex beads, processes that were blocked by a monoclonal antibody specific for TLR4. These data suggest that IFN-gamma primes neutrophils to respond to LPS.

Does physiological beta cell turnover initiate autoimmune diabetes in the regional lymph nodes?

The initial immune process that triggers autoimmune beta cell destruction in type 1 diabetes is not fully understood. In early infancy there is an increased beta cell turnover. Recurrent exposure of tissue-specific antigens could lead to primary sensitization of immune cells in the draining lymph nodes of the pancreas. An initial immune injury to the beta cells can be inflicted by several cell types, primarily macrophages and T cells. Subsequently, infiltrating macrophages transfer antigens exposed by apoptotic beta cells to the draining lymph nodes, where antigen presenting cells process and amplify a secondary immune reaction. Antigen presenting cells evolve as dual players in the activation and suppression of the autoimmune reaction in the draining lymph nodes. We propose a scenario where destructive insulitis is caused by recurrent exposure of specific antigens due to the physiological turnover of beta cells. This sensitization initiates the evolution of reactive clones that remain silent in the regional lymph nodes, where they succeed to evade regulatory clonal deletion.

The dual role of Fas-ligand as an injury effector and defense strategy in diabetes and islet transplantation.

The exact process that leads to the eruption of autoimmune reactions against beta cells and the evolution of diabetes is not fully understood. Macrophages and T cells may launch an initial immune reaction against the pancreatic islets of Langerhans, provoking inflammation and destructive insulitis. The information on the molecular mechanisms of the emergence of beta cell injury is controversial and points to possibly important roles for the perforin-granzyme, Fas-Fas-ligand (FasL) and tumor-necrosis-factor-mediated apoptotic pathways. FasL has several unique features that make it a potentially ideal immunomodulatory tool. Most important, FasL is selectively toxic to cytotoxic T cells and less harmful to regulatory T cells. This review discusses the intrinsic sensitivity of beta cells to FasL-mediated apoptosis, the conditions that underlie this beta cell sensitivity, and the feasibility of using FasL to arrest autoimmunity and prevent islet allograft rejection. In both the autoimmune and transplant settings, it is imperative to progress from the administration of nonspecific immunosuppressive therapy to the concept of beta-cell-specific immunomodulation. FasL evolves as a prime candidate for antigen-specific immunomodulation.

The p38 pathway partially mediates caspase-3 activation induced by reactive oxygen species in Fanconi anemia C cells.

Because Fanconi anemia (FA) cells display hypersensitivity to oxidative stress and reactive oxygen species (ROS) that act as second messengers in cellular signaling, we investigated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation in two FA-C lymphocyte cell lines (HSC536/N and PD149L) and one FA-A cell line (HSC99) exposed to interferon (IFN)-gamma or H2O2. IFN-gamma induced accumulation of ROS and activation of JNK and p38 in HSC536/N and PD149L but not in HSC99 cells. Higher concentrations of H2O2 were needed to induce moderate intracellular levels of ROS and phosphorylation of MAPKs in FA-A than in FA-C cells. Pre-incubation with dehydroascorbic acid resulted in reduced intracellular ROS levels and inhibition of MAPK activation induced by the above treatments. To define the functional role of JNK and p38 in IFN-gamma signaling, the effects of pharmacological inhibition of the MAPKs on induction of IFN-gamma and anti-Fas antibody responses were determined. Treatment of HSC536/N cells with p38-specific inhibitors partially inhibited caspase-3 activation while pre-incubation with specific inhibitors of JNK had no effect. Altogether, these results suggest that FA-C cells are hypersensitive to IFN-gamma and are more sensitive to oxidative stress than FA-A cells and that IFN-gamma and anti-Fas antibody mediate signals for apoptosis in FA-C cells via p38 but not JNK pathways.

An oxidative mechanism of interferon induced priming of the Fas pathway in Fanconi anemia cells.

Hematopoietic progenitor cells from children with Fanconi anemia of the C complementation group (FA-C) are excessively apoptotic and hypersensitive to various extracellular cues including Fas-ligand, tumor necrosis factor-alpha and double-stranded RNA. Interferon (IFN)-gamma is known to augment apoptotic responses of these factors. The "priming" effect of IFN-gamma is not fully explained. In view of the strong evidence that FA cells are intolerant of oxidative stress, we tested the notion that IFN-priming involves the induction of reactive oxygen species (ROS) in two FA-C B-lymphocyte cell lines and in peripheral blood neutrophils and mononuclear cells of FA patients. We also investigated whether the combination of IFN-gamma and Fas created an intracellular environment that promoted apoptosis. Significantly lower doses of IFN-gamma induced ROS accumulation in neutrophils and mononuclear cell of FA patients compared to cells of normal individuals. Enhanced ROS accumulation and decreased intracellular glutathione levels were observed in FA-C B-cell lines primed with IFN-gamma and treated with agonistic anti-Fas antibody than in isogenic control cells corrected with FANCC. The above treatment also induced caspase-3 and -8 activation as well as apoptosis. That antioxidants reduced the priming effect of IFN-gamma in Fas and IFN-gamma-treated FA lymphoblast cells, demonstrates that ROS represent a critical effector mechanism for the exaggerated responses to IFN-gamma characteristic of FA-C cells.