Marion Schneider

Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disease of early childhood characterized by nonmalignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes (macrophages). Cytotoxic T and natural killer (NK) cell activity is markedly reduced or absent in these patients, and mutations in a lytic granule constituent, perforin, were recently identified in a number of FHL individuals. Here, we report a comprehensive survey of 34 additional patients with FHL for mutations in the coding region of the perforin gene and the relative frequency of perforin mutations in FHL. Perforin mutations were identified in 7 of the 34 families investigated. Six children were homozygous for the mutations, and one patient was a compound heterozygote. Four novel mutations were detected: one nonsense, two missense, and one deletion of one amino acid. In four families, a previously reported mutation at codon 374, causing a premature stop codon, was identified, and, therefore, this is the most common perforin mutation identified so far in FHL patients. We found perforin mutations in 20% of all FHL patients investigated (7/34), with a somewhat higher prevalence, approximately 30% (6/20), in children whose parents originated from Turkey. No other correlation between the type of mutation and the phenotype of the patients was evident from the present study. Our combined results from mutational analysis of 34 families and linkage analysis of a subset of consanguineous families indicate that perforin mutations account for 20%-40% of the FHL cases and the FHL 1 locus on chromosome 9 for approximately 10%, whereas the major part of the FHL cases are caused by mutations in not-yet-identified genes.

Linkage of Familial Hemophagocytic Lymphohistiocytosis to 10q21-22 and Evidence for Heterogeneity

Familial hemophagocytic lymphohistiocytosis (FHL) is an autosomal recessive disorder characterized by the early onset of overwhelming activation of T lymphocytes and macrophages, invariably leading to death, in the absence of allogeneic bone marrow transplantation. Using genomewide genetic linkage analysis, we analyzed a group of 17 families with FHL and mapped a locus for FHL to the proximal region of the long arm of chromosome 10. Ten families showed no recombination with three tightly linked markers, D10S1650 (LOD score [Z]=6.99), D10S556 (Z=5.40), and D10S206 (Z=3.24), with a maximum multipoint LOD score of 11.22 at the D10S1650 locus. Haplotype analysis of these 10 families allowed us to establish D10S206 and D10S1665 as the telomeric and the centromeric flanking markers, respectively. Heterogeneity analysis and haplotype inspection of the remaining families confirmed that in seven families FHL was not linked to the 10q21-22 region, thus providing evidence for genetic heterogeneity of this condition.

Complement C3a and C5a modulate osteoclast formation and inflammatory response of osteoblasts in synergism with IL-1β

There is a tight interaction of the bone and the immune system. However, little is known about the relevance of the complement system, an important part of innate immunity and a crucial trigger for inflammation. The aim of this study was, therefore, to investigate the presence and function of complement in bone cells including osteoblasts, mesenchymal stem cells (MSC), and osteoclasts. qRT‐PCR and immunostaining revealed that the central complement receptors C3aR and C5aR, complement C3 and C5, and membrane‐bound regulatory proteins CD46, CD55, and CD59 were expressed in human MSC, osteoblasts, and osteoclasts. Furthermore, osteoblasts and particularly osteoclasts were able to activate complement by cleaving C5 to its active form C5a as measured by ELISA. Both C3a and C5a alone were unable to trigger the release of inflammatory cytokines interleukin (IL)‐6 and IL‐8 from osteoblasts. However, co‐stimulation with the pro‐inflammatory cytokine IL‐1β significantly induced IL‐6 and IL‐8 expression as well as the expression of receptor activator of nuclear factor‐kappaB ligand (RANKL) and osteoprotegerin (OPG) indicating that complement may modulate the inflammatory response of osteoblastic cells in a pro‐inflammatory environment as well as osteoblast–osteoclast interaction. While C3a and C5a did not affect osteogenic differentiation, osteoclastogenesis was significantly induced even in the absence of RANKL and macrophage‐colony stimulating factor (M‐CSF) suggesting that complement could directly regulate osteoclast formation. It can therefore be proposed that complement may enhance the inflammatory response of osteoblasts and increase osteoclast formation, particularly in a pro‐inflammatory environment, for example, during bone healing or in inflammatory bone disorders. J. Cell. Biochem. 112: 2594–2605, 2011.

Active Cytomegalovirus Infection in Patients with Septic Shock

Cytomegalovirus reactivation occurred in one third of patients and was associated with prolonged ventilation and stay in an intensive care unit.

Changes in the Novel Orphan, C5a Receptor (C5L2), during Experimental Sepsis and Sepsis in Humans

Sepsis is associated with extensive complement activation, compromising innate immune defenses, especially in neutrophils (PMN). Recently, a second C5a receptor (C5L2) was detected on PMN without evidence of intracellular signaling. The current study was designed to determine changes in C5L2 in blood PMN during sepsis. In vitro exposure of PMN to C5a, but not to fMLP, led to reduced content of C5L2. Following cecal ligation and puncture-induced sepsis in rats, PMN demonstrated a time-dependent decrease in C5L2. In vivo blockade of C5a during experimental sepsis resulted in preservation of C5L2. Similarly, PMN from patients with progressive sepsis showed significantly reduced C5L2 expression (n = 26), which was virtually abolished in patients who developed multiorgan failure (n = 10). In contrast, sepsis survivors exhibited retention of C5L2 (n = 12/13). The data suggest that C5L2 on PMN diminishes during sepsis due to systemic generation of C5a, which is associated with a poor prognosis.

Trauma-Induced Systemic Inflammatory Response versus Exercise-Induced Immunomodulatory Effects

Accidental trauma and heavy endurance exercise, both induce a kind of systemic inflammatory response, also called systemic inflammatory response syndrome (SIRS). Exercise-related SIRS is conditioned by hyperthermia and concomitant heat shock responses, whereas trauma-induced SIRS manifests concomitantly with tissue necrosis and immune activation, secondarily followed by fever. Inflammatory cytokines are common denominators in both trauma and exercise, although there are marked quantitative differences. Different anti-inflammatory cytokines may be involved in the control of inflammation in trauma- and exercise-induced stress. Exercise leads to a balanced equilibrium between inflammatory and anti-inflammatory responses. Intermittent states of rest, as well as anti-oxidant capacity, are lacking or minor in trauma but are high in exercising individuals. Regular training may enhance immune competence, whereas trauma-induced SIRS often paves the way for infectious complications, such as sepsis.

Cellular Immunity and Active Human Cytomegalovirus Infection in Patients with Septic Shock

BACKGROUND Human cytomegalovirus (CMV) is an important opportunistic pathogen after transplantations. In the present study, monitoring of CMV in patients with septic shock was used to discover whether T helper cell type 1 (Th1) cell and natural killer (NK) cell functions interact with CMV reactivation in patients not undergoing immunosuppressive therapy. METHODS Thirty-eight patients with septic shock were monitored, and the 23 CMV-seropositive patients were included in this prospective study. RESULTS Seven patients (30.4%) developed an active CMV infection despite the detection of CMV-reactive Th1 cells. After active CMV infection, the frequency of CMV-reactive Th1 cells increased from a median of 0.52% to 5.04% (P=.009). Active CMV infections were terminated without antiviral therapy within 2 weeks. In parallel, the frequency of staphylococcal enterotoxin B (SEB; superantigen)-reactive Th1 cells increased from a median of 1.11% to 8.48% (P=.027). In patients without active CMV infection, the frequency of CMV-reactive (median, 0.39%) and SEB-reactive (median, 1.11%) Th1 cells did not increase. Cytotoxic NK cell activity was persistently suppressed despite the presence of CD56(+)CD16(+) NK cells. Moreover, interleukin-2 application in vitro did not restore NK cell activity. CONCLUSIONS A proinflammatory immune response may contribute to CMV reactivation in patients with septic shock. Adaptive T cell immunity, more likely than NK cell immunity, may contribute to termination of active CMV infection without antiviral therapy in these patients.

Anticipatory Symptoms and Anticipatory Immune Responses in Pediatric Cancer Patients Receiving Chemotherapy: Features of a Classically Conditioned Response?

UNLABELLED There is considerable evidence from studies in adult patients that classical conditioning contributes to anticipatory nausea and/or vomiting (ANV) in cancer chemotherapy: The stimuli predicting the infusion serve as conditioned stimuli (CS). When reexposed to the CS, some patients experience ANV prior to infusion onset. In adult patients, anticipatory immunomodulation (AIM) has also been observed. The present study examines whether ANV and AIM occur in pediatric cancer patients and whether they show features of a conditioned response. METHODS Nineteen pediatric cancer patients (M = 10.1 years, > 2 previous chemotherapies) were studied over two consecutive cycles (A, B). In both cycles, self-reported symptoms, for example nausea and vomiting, were recorded from two days prior to the onset (Day -2), during infusion, and two days after the end of the infusion (Day +2). In Cycle B, blood was drawn at home at Day -2, and at Day 0 in the hospital prior to infusion onset, thus using a quasi-experimental variation of the CS content of the environment. Immune parameters valid for tumor defense and cytotoxic competence (natural killer cell activity [NKCA], plasma interleukin [IL]-1beta, IL-2, IL-10, interferon [IFN]-gamma, tumor necrosis factor [TNF]-alpha) and cortisol were measured. RESULTS ANV was reported by 7 patients in at least one cycle. In Cycle A, ANV was positively associated with emetogenity of chemotherapy. Features of ANV-duration and occurrence-tended to be positively associated with those of posttreatment nausea and vomiting. AN increased as infusion onset time approached. NKCA and IFN-gamma increased from home to hospital, independent from cortisol level. The NKCA increase was predominantly observed in patients with ANV. CONCLUSIONS ANV in pediatric patients showed features of a CR. Immune parameters were sensitive to the CS content of the environment, predominantly in patients with ANV. This is consistent with the manifestation of multiple CRs.

Changes and regulation of the C5a receptor on neutrophils during septic shock in humans.

During experimental sepsis, excessive generation of the anaphylatoxin C5a results in reduction of the C5a receptor (C5aR) on neutrophils. These events have been shown to result in impaired innate immunity. However, the regulation and fate of C5aR on neutrophils during sepsis are largely unknown. In contrast to 30 healthy volunteers, 60 patients in septic shock presented evidence of complement activation with significantly increased serum levels of C3a, C5a, and C5b-9. In the septic shock group, the corresponding decrease in complement hemolytic activity distinguished survivors from nonsurvivors. Neutrophils from patients in septic shock exhibited decreased C5aR expression, which inversely correlated with serum concentrations of C-reactive protein (CRP) and clinical outcome. In vitro exposure of normal neutrophils to native pentameric CRP led to a dose- and time-dependent loss of C5aR expression on neutrophils, whereas the monomeric form of CRP, as well as various other inflammatory mediators, failed to significantly alter C5aR levels on neutrophils. A circulating form of C5aR (cC5aR) was detected in serum by immunoblotting and a flow-based capture assay, suggestive of an intact C5aR molecule. Levels of cC5aR were significantly enhanced during septic shock, with serum levels directly correlating with lethality. The data suggest that septic shock in humans is associated with extensive complement activation, CRP-dependent loss of C5aR on neutrophils, and appearance of cC5aR in serum, which correlated with a poor outcome. Therefore, cC5aR may represent a new sepsis marker to be considered in tailoring individualized immune-modulating therapy.

Different patient case mix by applying the 2003 SCCM/ESICM/ACCP/ATS/SIS sepsis definitions instead of the 1992 ACCP/SCCM sepsis definitions in surgical patients: a retrospective observational study

BackgroundRevised consensus sepsis definitions have been published in 2003. The present study was performed to compare the prevalence of different stages of sepsis and ICU mortality rates and find out the case mix within the same collective of postoperative/posttraumatic patients applying either the original 1992 ACCP/SCCM or the revised 2003 SCCM/ESICM/ACCP/ATS/SIS sepsis definitions.MethodsRetrospective observational single-centre study in surgical critically ill patients admitted to an University adult ICU. From 01/2007 to 12/2007, 742 patients were surveyed daily computer-assisted with respect to different stages of sepsis.ResultsWithin the same patient collective, applying the 2003 definitions instead of the 1992 definitions, prevalence of severe sepsis (61 vs. 56) and septic shock (205 vs. 162) was higher (p < 0.001). In patients with septic shock according to the 2003 definitions, mortality rate of 22% was lower than that of 27%, when the 1992 definitions were used. Risk of death was increased for those patients classified to be in septic shock with any of the definitions (OR 6.5, p = 0.001). Sensitivity to predict deaths was slightly higher with the 2003 definitions (92%) than with the 1992 definitions (88%), and specificity was lower (31% vs. 49%).ConclusionThe prevalence and mortality rates of various sepsis severity stages differ if defined by the 1992 or the 2003 definitions. Thus, transferring conclusions drawn from data sets regarding severity of sepsis generated with the 1992 definitions to the same population applying the 2003 definitions may be misleading. The 1992 definitions may under-classify patients with severe sepsis.