Decreased myoblast differentiation in chronic binge alcohol-administered simian immunodeficiency virus-infected male macaques: role of decreased miR-206.

Skeletal muscle stem cells play a critical role in regeneration of myofibers. We previously demonstrated that chronic binge alcohol (CBA) markedly attenuates myoblast differentiation potential and myogenic gene expression. Muscle-specific microRNAs (miRs) are implicated in regulation of myogenic genes. The aim of this study was to determine whether myoblasts isolated from asymptomatic CBA-administered simian immunodeficiency virus (SIV)-infected macaques treated with antiretroviral therapy (ART) showed similar impairments and, if so, to elucidate potential underlying mechanisms. Myoblasts were isolated from muscle at 11 mo after SIV infection from CBA/SIV macaques and from time-matched sucrose (SUC)-treated SIV-infected (SUC/SIV) animals and age-matched controls. Myoblast differentiation and myogenic gene expression were significantly decreased in myoblasts from SUC/SIV and CBA/SIV animals compared with controls. SIV and CBA decreased muscle-specific miR-206 in plasma and muscle and SIV decreased miR-206 expression in myoblasts, with no statistically significant changes in other muscle-specific miRs. These findings were associated with a significant increase in histone deacetylase 4 (HDAC4) and decrease in myogenic enhancer factor 2C (MEF2C) expression in CBA/SIV muscle. Transfection with miR-206 inhibitor decreased myotube differentiation, increased expression of HDAC4, and decreased MEF2C, suggesting a critical role of miR-206 in myogenesis. Moreover, HDAC4 was confirmed to be a direct miR-206 target. These results support a mechanistic role for decreased miR-206 in suppression of myoblast differentiation resulting from chronic alcohol and SIV infection. The parallel changes in skeletal muscle and circulating levels of miR-206 warrant studies to establish the possible use of plasma miR-206 as an indicator of impaired muscle function.

Immune response gene profiles in the term placenta depend upon maternal muscle mass.

Maternal thinness leads to metabolic challenges in the offspring, but it is unclear whether reduced maternal fat mass or muscle mass drives these metabolic changes. Recently, it has been shown that low maternal muscle mass--as measured by arm muscle area (AMA)--is associated with depressed nutrient transport to the fetus. To determine the role of maternal muscle mass on placental function, we analyzed the gene expression profiles of 30 human placentas over the range of AMA (25.2-90.8 cm(2)) from uncomplicated term pregnancies from the Southampton Women's Survey cohort. Eighteen percent of the ∼60 genes that were highly expressed in less muscular women were related to immune system processes and the interferon-γ (IFNG) signaling pathway in particular. Those transcripts related to the IFNG pathway included IRF1, IFI27, IFI30, and GBP6. Placentas from women with low muscularity are, perhaps, more sensitive to the effects of inflammatory cytokines than those from more muscular women.

Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression.

Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis.

Ulcerative colitis presenting as purpura fulminans.

A 60-year-old man presented with purpura fulminans involving his chest and flank. He was subsequently found to have active ulcerative colitis (UC) and protein S deficiency. He was treated with heparin and plasma, but because of persistent colitis and progressively worsening purpura, a total colectomy was performed on hospital day 17. This report describes an interesting case of purpura fulminans associated with the hypercoagulable state of active UC that responded dramatically to colectomy.

Increased granulopoiesis through interleukin-17 and granulocyte colony-stimulating factor in leukocyte adhesion molecule-deficient mice.

Many mutant mice deficient in leukocyte adhesion molecules display altered hematopoiesis and neutrophilia. This study investigated whether peripheral blood neutrophil concentrations in these mice are elevated as a result of accumulation of neutrophils in the circulation or altered hematopoiesis mediated by a disrupted regulatory feedback loop. Chimeric mice were generated by transplanting various ratios of CD18(+/+) and CD18(-/-) unfractionated bone marrow cells into lethally irradiated wild-type mice, resulting in approximately 0%, 10%, 50%, 90%, or 100% CD18 null neutrophils in the blood. The presence of only 10% CD18(+/+) neutrophils was sufficient to prevent the severe neutrophilia seen in mice reconstituted with CD18(-/-) bone marrow cells. These data show that the neutrophilia in CD18(-/-) mice is not caused by enhanced neutrophil survival or the inability of neutrophils to leave the vascular compartment. In CD18(-/-), CD18(-/-)E(-/-), CD18(-/-)P(-/-), EP(-/-), and EPI(-/-) mice, levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-17 (IL-17) were elevated in proportion to the neutrophilia seen in these mice, regardless of the underlying mutation. Antibiotic treatment or the propensity to develop skin lesions did not correlate with neutrophil counts. Blocking IL-17 or G-CSF function in vivo significantly reduced neutrophil counts in severely neutrophilic mice by approximately 50% (P <.05) or 70% (P <.01), respectively. These data show that peripheral blood neutrophil numbers are regulated by a feedback loop involving G-CSF and IL-17 and that this feedback loop is disrupted when neutrophils cannot migrate into peripheral tissues.

Acute ethanol intoxication suppresses lung chemokine production following infection with Streptococcus pneumoniae.

Alcohol intoxication impairs neutrophil function and increases host susceptibility to Streptococcus pneumoniae. In a rat model of pneumonia, the effects of acute intoxication were monitored for lung chemokine responses, neutrophil recruitment, and bactericidal activity. Alcohol delayed lung neutrophil recruitment, increased bacterial burden, and decreased survival. Before neutrophil recruitment, bronchoalveolar lavage (BAL) macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) were decreased by alcohol. This alcohol-induced effect was reversed at 6 h, when there were large numbers of neutrophils in control BAL fluid, compared with the alcohol-treated group. Cyclophosphamide-induced neutropenia decreased neutrophil recruitment, minimizing the effects of recruited neutrophils on chemokine levels, and extended the alcohol-induced chemokine suppression. MIP-2 and CINC mRNA contents also were suppressed by alcohol 4 and 6 h after infection. Thus, alcohol suppresses lung chemokine activity in response to S. pneumoniae, which is associated with delayed neutrophil delivery, elevated bacterial burden, and increased mortality.

Interleukin-17 and lung host defense against Klebsiella pneumoniae infection.

Bacterial pneumonia remains an important cause of morbidity and mortality worldwide, especially in immune-compromised patients. Cytokines and chemokines are critical molecules expressed in response to invading pathogens and are necessary for normal lung bacterial host defenses. Here we show that interleukin (IL)-17, a novel cytokine produced largely by CD4+ T cells, is produced in a compartmentalized fashion in the lung after challenge with Klebsiella pneumoniae. Moreover, overexpression of IL-17 in the pulmonary compartment using a recombinant adenovirus encoding murine IL-17 (AdIL-17) resulted in the local induction of tumor necrosis factor-alpha, IL-1beta, macrophage inflammatory protein-2, and granulocyte colony-stimulating factor (G-CSF); augmented polymorphonuclear leukocyte recruitment; and enhanced bacterial clearance and survival after challenge with K. pneumoniae. However, simultaneous treatment with AdIL-17 provided no survival benefit after intranasal K. pneumoniae challenge. These data show that IL-17 may have a role in priming for enhanced chemokine and G-CSF production in the context of lung infection and that optimally timed gene therapy with IL-17 may augment host defense against bacterial pneumonia.

The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality.

The Fanconi anemia (FA) group C gene product (FANCC) functions to protect cells from cytotoxic and genotoxic effects of cross-linking agents. FANCC is also required for optimal activation of STAT1 in response to cytokine and growth factors and for suppressing cytokine-induced apoptosis by modulating the activity of double-stranded RNA-dependent protein kinase. Because not all FANCC mutations affect STAT1 activation, the hypothesis was considered that cross-linker resistance function of FANCC depends on structural elements that differ from those required for the cytokine signaling functions of FANCC. Structure-function studies were designed to test this notion. Six separate alanine-substituted mutations were generated in 3 highly conserved motifs of FANCC. All mutants complemented mitomycin C (MMC) hypersensitive phenotype of FA-C cells and corrected aberrant posttranslational activation of FANCD2 in FA-C mutant cells. However, 2 of the mutants, S249A and E251A, failed to correct defective STAT1 activation. FA-C lymphoblasts carrying these 2 mutants demonstrated a defect in recruitment of STAT1 to the interferon gamma (IFN-gamma) receptor and GST-fusion proteins bearing S249A and E251A mutations were less efficient binding partners for STAT1 in stimulated lymphoblasts. These same mutations failed to complement the characteristic hypersensitive apoptotic responses of FA-C cells to tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma. Cells bearing a naturally occurring FANCC mutation (322delG) that preserves this conserved region showed normal STAT1 activation but remained hypersensitive to MMC. The conclusion is that a central highly conserved domain of FANCC is required for functional interaction with STAT1 and that structural elements required for STAT1-related functions differ from those required for genotoxic responses to cross-linking agents. Preservation of signaling capacity of cells bearing the del322G mutation may account for the reduced severity and later onset of bone marrow failure associated with this mutation.

FANCC interacts with Hsp70 to protect hematopoietic cells from IFN-gamma/TNF-alpha-mediated cytotoxicity.

The Fanconi anemia (FA) complementation group C gene product (FANCC) functions to protect hematopoietic cells from cytotoxicity induced by interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and double-stranded RNA (dsRNA). Because apoptotic responses of mutant FA-C cells involve activation of interferon-inducible, dsRNA-dependent protein kinase PKR, we sought to identify FANCC-binding cofactors that may modulate PKR activation. We identified the molecular chaperone Hsp70 as an interacting partner of FANCC in lymphoblasts and HeLa cells using 'pull-down' and co-immunoprecipitation experiments. In vitro binding assays showed that the association of FANCC and Hsp70 involves the ATPase domain of Hsp70 and the central 320 residues of FANCC, and that both Hsp40 and ATP/ADP are required. In whole cells, Hsp70-FANCC binding and protection from IFN-gamma/TNF-alpha-induced cytotoxicity were blocked by alanine mutations located in a conserved motif within the Hsp70-interacting domain of FANCC. We therefore conclude that FANCC acts in concert with Hsp70 to prevent apoptosis in hematopoietic cells exposed to IFN-gamma and TNF-alpha.

Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.

Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.

Functional correction of FA-C cells with FANCC suppresses the expression of interferon gamma-inducible genes.

Because hematopoietic cells derived from Fanconi anemia (FA) patients of the C-complementation group (FA-C) are hypersensitive to the inhibitory effects of interferon gamma (IFNgamma), the products of certain IFNgamma-inducible genes known to influence hematopoietic cell survival were quantified. High constitutive expression of the IFNgamma-inducible genes, IFN-stimulated gene factor 3 gamma subunit (ISGF3gamma), IFN regulatory factor-1 (IRF-1), and the cyclin-dependent kinase inhibitor p21(WAF1) was found in FANCC mutant B lymphoblasts, low-density bone marrow cells, and murine embryonic fibroblasts. Paradoxically, these cells do not activate signal transducer and activator of transcription (STAT) 1 properly. In an attempt to clarify mechanisms by which FA-C cells overexpress IFNgamma-inducible genes in the face of defective STAT1 phosphorylation, it was reasoned that decreased levels of activated STAT1 might result in reduced expression of a hematopoietic IFNgamma-responsive protein that normally modulates expression of other IFNgamma-responsive genes. Levels of the IFNgamma-inducible factor IFN consensus sequence binding protein (ICSBP), a negative trans-acting regulator of some IFNgamma-inducible genes, were quantified. ICSBP levels were reduced in FA-C B lymphoblasts and MEFs. However, enforced expression of ICSBP failed to down-regulate IRF-1, ISGF3gamma, and p21(WAF1). Thus, the FANCC protein functions to modulate expression of a family of genes that in normal cells are inducible only by specific environmental cues for apoptosis or mitogenic inhibition, but it does so independently of the classic IFN-STAT1 pathway and is not the direct result of reduced ICSBP expression.

Prolonged ethanol treatment enhances lipopolysaccharide/phorbol myristate acetate-induced tumor necrosis factor-alpha production in human monocytic cells.

BACKGROUND: Ethanol (EtOH) is known to alter host immune responses and cytokine production. Acute EtOH exposure can suppress tumor necrosis factor (TNF)-alpha production, which attenuates pulmonary defense against infection. Previous studies in our laboratory show that acute EtOH inhibited TNF-alpha production by a posttranscriptional process, namely suppression of TNF-alpha-converting, enzyme-mediated, ectodomain shedding. However, chronic EtOH has been shown to augment TNF-alpha production, and this has been associated with EtOH-induced liver injury. To further characterize this paradoxical effect of EtOH on TNF-alpha production, we developed an in vitro model by using Mono Mac 6 cells, a human monocytic cell line. METHODS: Mono Mac 6 cells were treated with EtOH (0-75 mM) for 1 to 7 days. TNF-alpha production was induced by lipopolysaccharide and phorbol myristate acetate and quantitated by enzyme-linked immunosorbent assay. Generation of reactive oxygen species (ROS) was assayed by using a specific fluorogenic reagent. RESULTS: Acute EtOH initially inhibited lipopolysaccharide/phorbol myristate acetate-induced TNF-alpha production in Mono Mac 6 cells. However, during chronic EtOH exposure, this inhibition was reversed gradually over time. By day 6 after EtOH treatment, Mono Mac 6 cells demonstrated significant up-regulation of TNF-alpha production. Moreover, chronic EtOH induced the generation of ROS in these Mono Mac 6 cells. Scavenging ROS by Mn(III)tetrakis(1-methyl-4pyridyl)porphyrin pentachloride and N-acetyl-L-cysteine attenuated chronic EtOH-enhanced TNF-alpha production. CONCLUSION: These results suggest that ROS induction is involved in EtOH-enhanced TNF-alpha production by monocytes. This study also provides insight into the mechanisms of alteration of TNF-alpha production in different EtOH exposure settings.

Cytokine treatment of bacterial pneumonia.

The lungs represent the largest epithelial surface area of the body and are repeatedly exposed to a variety of potential pathogens. The respiratory system has, therefore, developed an elaborate system of defense mechanisms to maintain the sterility of the lower airways. In the event of a defect, either developmental or acquired of any component of these defenses, the host is rendered susceptible to infection of the respiratory tract. Conventional therapy primarily focuses on the selection of appropriate antibiotic therapy and institution of supportive measures. In this article, we discuss emerging immune-based strategies in the treatment of pneumonia that are primarily focused on amplification of the host defense system through the administration of cytokines.

Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon gamma, tumor necrosis factor-alpha, and double-stranded RNA.

Hematopoietic cells bearing inactivating mutations of Fanconi anemia group C (FANCC) are excessively apoptotic and demonstrate hypersensitivity not only to cross-linking agents but also to interferon gamma (IFN-gamma) and tumor necrosis factor-alpha. Seeking essential signaling pathways for this phenotype, this study quantified constitutive and induced RNA-dependent protein kinase (PKR) activation in Fanconi anemia cells of the C complementation group (FA-C). PKR was constitutively phosphorylated and exhibited an increased binding affinity for double-stranded RNA (dsRNA) in FANCC(-/-) cells. FANCC(-/-) cells were hypersensitive to both dsRNA and the combination of dsRNA and IFN-gamma in that these agents induced a higher fraction of apoptosis in FANCC(-/-) cells than in normal cells. Overexpression of wild-type PKR-sensitized FANCC(-/-) cells to apoptosis induced by IFN-gamma and dsRNA. Conversely, inhibition of PKR function by enforced expression of a dominant-negative inhibitory mutant of PKR (PKRDelta6) substantially reduced the IFN and dsRNA hypersensitivity of FANCC(-/-) cells. Two PKR target molecules, IkappaB-alpha and IRF-1, were not differentially activated in FANCC(-/-) cells, but enforced expression of a nonphosphorylatable form of eukaryotic translation initiation factor-2alpha reversed the PKR-mediated block of messenger RNA translation and partially abrogated the PKR-mediated apoptosis in FANCC(-/-) cells. Because no evidence was found of a PKR/FANCC complex in normal cells, it was concluded that an essential function of FANCC is to suppress, indirectly, the activity of PKR and that FANCC inactivation results in IFN hypersensitivity, at least in part, because this function of FANCC is abrogated.

The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin.

Although G-CSF has been shown to increase neutrophil (polymorphonuclear leukocyte, PMN) recruitment into the lung during pulmonary infection, relatively little is known about the local chemokine profiles associated with this enhanced PMN delivery. We investigated the effects of G-CSF and PMN recruitment on the pulmonary chemokine response to intratracheal LPS. Rats pretreated twice daily for 2 days with an s.c. injection of G-CSF (50 microg/kg) were sacrificed at either 90 min or 4 h after intratracheal LPS (100 microg) challenge. Pulmonary recruitment of PMNs was not observed at 90 min post LPS challenge. Macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) concentrations in bronchoalveolar lavage (BAL) fluid were similar in animals pretreated with or without G-CSF at this time. G-CSF pretreatment enhanced pulmonary recruitment of PMNs (5-fold) and greatly reduced MIP-2 and CINC levels in BAL fluid at 4 h after LPS challenge. In vitro, the presence of MIP-2 and CINC after LPS stimulation of alveolar macrophages was decreased by coculturing with circulating PMNs but not G-CSF. G-CSF had no direct effect on LPS-induced MIP-2 and CINC mRNA expression by alveolar macrophages. Pulmonary recruited PMNs showed a significant increase in cell-associated MIP-2 and CINC. Cell-associated MIP-2 and CINC of circulating PMNs were markedly increased after exposure of these cells to the BAL fluid of LPS-challenged lungs. These data suggest that recruited PMNs are important cells in modulating the local chemokine response. G-CSF augments PMN recruitment and, thereby, lowers local chemokine levels, which may be one mechanism resulting in the subsidence of the host proinflammatory response.

Hemorrhage alters neuroendocrine, hemodynamic, and compartment-specific TNF responses to LPS.

The aim of the present study was to determine the effects of fixed pressure (40 mmHg) hemorrhage (HEM) followed by fluid resuscitation with Ringer's lactate on the subsequent hemodynamic, neurohormonal, and TNF response elicited by systemic lipopolysaccharide (LPS) administration. Chronically catheterized, conscious, unrestrained male Sprague-Dawley rats were randomized to either HEM (n = 12) or sham (n = 12) groups. HEM and sham animals were randomized to receive either LPS (100 mg/100 g body weight) or an equal volume of intravenous saline 1.5 h after completion of the resuscitation period. LPS administration produced an immediate 20% decrease in mean arterial pressure in sham animals, which was accentuated in HEM animals (40%, P < 0.05 versus sham). Moreover, HEM blunted (75%, P < 0.05) the LPS-induced increase in plasma TNF concentrations. TNF was not detected in bronchoalveolar lavage fluid (BALF) obtained from sham LPS-treated animals. In contrast, TNF levels were significantly elevated (35 +/- 17 pg/mL) in HEM LPS-treated animals. A 400% increase in lung TNF content following LPS treatment was not affected by prior HEM. LPS administration produced a marked increase in plasma epinephrine, norepinephrine, and corticosterone levels in sham animals. HEM blunted the LPS-induced rise in circulating levels of epinephrine and corticosterone without altering that of norepinephrine. Our second set of studies showed that the increase in BALF TNF was associated with a 30% increase in wet-to-dry lung weight ratios, suggesting that this is most likely the result of leaky endothelium following hemorrhage and LPS. Furthermore, alterations in LPS-induced alveolar macrophage TNF production following HEM were not detected. These results indicate that HEM altered the hemodynamic, neurohormonal, and circulating TNF responses to systemic LPS administration. In addition, our results suggest that HEM impaired the compartmentalization of the inflammatory response to LPS, without affecting alveolar macrophage responses to LPS. The role of altered neuroendocrine responses to a second challenge in modulating proinflammatory responses remains to be elucidated.

Severe inflammatory defect and reduced viability in CD18 and E-selectin double-mutant mice.

CD18-deficient mice (CD18(-/-) mice) have a severe leukocyte recruitment defect in some organs, and no detectable defect in other models. Mice lacking E-selectin (CD62E(-/-) mice) have either no defect or a mild defect of neutrophil infiltration, depending on the model. CD18(-/-)CD62E(-/-), but not CD18(-/-)CD62P(-/-), mice generated by crossbreeding failed to thrive, reaching a maximum body weight of 10-15 grams. To explore the mechanisms underlying reduced viability, we investigated lethally irradiated CD62E(-/-) mice that were reconstituted with CD18(-/-) bone marrow. These mice, but not single-mutant controls, showed tenfold-increased rolling velocities in a TNF-alpha-induced model of inflammation. Leukocyte adhesion efficiency in CD18(-/-)CD62E(-/-) mice was reduced by 95%, and hematopoiesis was drastically altered, including severe bone marrow and blood neutrophilia and elevated G-CSF and GM-CSF levels. The greatly reduced viability of CD18(-/-)CD62E(-/-) mice appears to result from an inability to mount an adequate inflammatory response. Our data show that cooperation between E-selectin and CD18 integrins is necessary for neutrophil recruitment and that alternative adhesion pathways cannot compensate for the loss of these molecules.

Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members.

Hematopoietic progenitor cells (HPC) from mice nullizygous at the Fanconi anemia (FA) group C locus and children with Fanconi anemia group C (FA-C) are hypersensitive to interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. This hypersensitivity results, in part, from the capacity of these cytokines to prime the fas pathway. Because fas-mediated programmed cell death in many cells involves sequential activation of specific caspases, we tested the hypothesis that programmed cell death in FA HPC involves the ordered activation of specific caspase molecules. Lysates from lymphoblasts treated with both agonistic anti-fas antibody and IFN-gamma contained activated caspase 3 family members (caspases 3, 6, and 7), as well as caspase 8, whereas activation of caspases 1, 2, 4, 9, and 10 was not detected. The apoptotic effects of fas agonists in IFN-gamma-treated human and murine FA-C cells were blocked when pretreated with inhibitors (ac-DEVD-cho, CP-DEVD-cho, Z-DEVD-FMK) of the caspase 3 protease. Inhibitors (ac-YVAD-cho, CP-YVAD-cho, Z-YVAD-FMK) of caspase 1 did not block apoptosis or caspase 3 activation. Treatment of FA cells with the fluoromethyl ketone tetrapeptide caspase 8 inhibitor (ac-IETD-FMK) did suppress caspase 3 activation. A 4-fold greater fraction of IFN-induced FA-C cells expressed caspase 3 than FA-C cells complemented by retroviral-mediated transfer of FANCC. Therefore fas-induced apoptosis in Fanconi anemia cells of the C type involves the activation of caspase 8, which controls activation of caspase 3 family members and one direct or indirect function of the FANCC protein is to suppress apoptotic responses to IFN-gamma upstream of caspase 3 activation. (Blood. 2000;96:4204-4211)

Monitoring patients taking oral carbonic anhydrase inhibitors.

Although no prospective studies have been done and no recommendations can be reasonably evidenced based, we believe that our "alternative views" are reason-able, taking into account the serious nature of aplastic anemia and its more favorable prognosis when this disorder is treated early. Because aplastic anemia represents only about one half of possible CAl blood dyscrasias, early recognition of non-aplastic anemia dyscrasias alone Justifies hematologic screening.