Delayed Severe Hemolytic Transfusion Reaction During Pregnancy in a Woman with ß-Thalassemia Intermediate: Successful Outcome After Eculizumab Administration.

BACKGROUND Delayed hemolytic transfusion reactions (DHTR) are life-threatening complications mostly triggered by red blood cell (RBC) transfusions in patients with hemoglobinopathy. CASE REPORT We present a case of DHTR and hyperhemolysis syndrome in a 39-year-old pregnant woman with a history of ß-thalassemia intermediate in whom the hemoglobin (Hb) level fell to 27 g/L after transfusion of 2 units of crossmatch-compatible packed RBCs. No allo- or auto-antibody formation was detected. Administration of intravenous immunoglobulins and methylprednisolone followed by anti-CD20 rituximab was tried, but was unsuccessful. Infusions of eculizumab (900 mg twice at a 7-day interval) followed by another course of intravenous immunoglobulins (2 g/kg/day for 5 days) and combined with repeated erythropoietin injections (darbepoetin alpha 300 µg/week) finally allowed biological and clinical improvement. Blood counts remained controlled until delivery. Despite signs of intrauterine growth retardation, she gave birth by cesarean section at 31 weeks of pregnancy to a 1.15-kg infant. CONCLUSIONS Eculizumab seems to be of benefit in DHTR associated with hyperhemolysis and should be used early in the treatment of this pathology. Despite premature birth, our case report showed an acceptable outcome for the infant when eculizumab treatment was used during pregnancy.

Decreased monocyte human leukocyte antigen-DR expression after severe burn injury: Correlation with severity and secondary septic shock.

OBJECTIVE: Severe thermal injury causes immune dysfunctions involving both pro- and anti-inflammatory mechanisms. It subsequently leads to a state of immune deficiency that shares some similarities with sepsis-induced immunosuppression. A hallmark of the latter is established by decreased monocyte human leukocyte antigen-DR (mHLA-DR) measurements. The main objective of the current study was to characterize the appearance and the duration of low mHLA-DR expression after severe burn as well as to determine its correlation with mortality and septic complications. DESIGN: Observational study. SETTING: Burn unit (intensive care unit) in a university hospital. PATIENTS: Severe burn patients (total burn surface area >30%, n = 14) and healthy individuals (n = 29). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients were immunologically monitored during 15 days. We quantified mHLA-DR expression with a standardized flow cytometry protocol. Every patient presented with decreased mHLA-DR expression at days 2-3 after burn. Then, from days 4-6, this expression increased in patients who would survive whereas it remained low in nonsurvivors. As early as days 7-10 after burn, patients who were going to develop secondary septic shock exhibited significantly lower mHLA-DR expression in comparison with patients recovering without severe septic complications. Using quantitative reverse transcriptase-polymerase chain reaction, at days 4-6, we found that the RNA level of the nonpolymorphic HLA-DRA chain and the transcription factor class II transactivator were also significantly decreased compared with healthy controls; however, plasma cytokines (interleukin-6, tumor necrosis factor-alpha, and interleukin-10) did not provide any significant prognostic information. CONCLUSIONS: Severe burn injury induced a marked reduction in mHLA-DR expression at both protein and messenger RNA levels. Its persistent decrease was associated with mortality and the development of septic complications.

Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism.

Although it is known that septic shock induces immunosuppression, the mechanism for this phenomenon is not well understood. Monocytes play a central role in septic shock pathophysiology, which is also characterized by an increased proportion of natural regulatory T (Treg) cells. We therefore investigated whether Treg could be involved in the decreased monocyte expression of CD14 and HLA-DR observed during septic shock. We demonstrated that human Treg inhibit LPS-induced retention of monocyte CD14. Because loss of CD14 is a hallmark of monocyte apoptosis, this suggests that Treg inhibit monocyte survival. This effect was largely mediated through the release of a soluble mediator that was not identical with either IL-10 or IL-4. The Fas/FasL pathway participated in the effect as it was blocked by anti-FasL Abs and reproduced by Fas agonist and recombinant soluble FasL. Furthermore, expression of FasL was much higher on Treg than on their CD25(-) counterparts. Collectively, these results indicate that Treg act on monocytes by inhibiting their LPS-induced survival through a proapoptotic mechanism involving the Fas/FasL pathway. This may be an important mechanism for septic shock-induced immunosuppression and may offer new perspectives for the treatment of this deadly disease.

Comparative inflammatory properties of staphylococcal superantigenic enterotoxins SEA and SEG: implications for septic shock.

The severity of Staphylococcus aureus sepsis is positively associated with staphylococcal enterotoxin A (SEA) and negatively associated with the enterotoxin gene cluster (egc), which encodes five staphylococcal enterotoxins. We postulated that the variable, clinical severity of S. aureus sepsis might be a result of differences in the inflammatory properties of staphylococcal superantigens. We therefore compared the inflammatory properties of SEA with those of staphylococcal entérotoxin G (SEG), a member of the five egc superantigens. We found that SEA and SEG had similar superantigenic properties, as they induced CD69 expression on T lymphocytes and selective expansion of Vbeta subpopulations. Contrary to SEG, however, SEA induced a strong proinflammatory/Th1 response, including TNF-alpha and MIP-1alpha production. These results suggest that the association of SEA with the severity of S. aureus septic shock, characterized by a deleterious, inflammatory cascade, may be explained partly by the specific proinflammatory properties of this superantigen.

Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock.

OBJECTIVE: The immediate overwhelming release of inflammatory mediators in septic shock is rapidly followed by strong anti-inflammatory responses inducing a state of immunosuppression. The patients who survive the initial hyper-inflammatory step of septic shock but subsequently die may be those who do not recover from immunosuppression. We assessed whether a low monocyte human leukocyte antigen-DR (mHLA-DR) expression, proposed as a marker of immunosuppression, is an independent predictor of mortality in patients who survived the initial 48 h of septic shock. DESIGN AND SETTING: Prospective observational study performed in two adult intensive care units at a university hospital. PATIENTS: 93 consecutive patients with septic shock. MEASUREMENTS AND RESULTS: At days 1-2, mHLA-DR values (determined by flow cytometry) were not significantly different between survivors and non-survivors. A sharp difference became highly significant at days 3-4 when survivors had increased their values, while non-survivors had not (43% vs. 18%, percentage of HLA-DR positive monocyte, p < 0.001). Multivariate logistic regression analysis revealed that low mHLA-DR (< 30%) at days 3-4 remained independently associated with mortality after adjustment for usual clinical confounders, adjusted odds ratio (CI): 6.48 (95% CI: 1.62-25.93). CONCLUSION: The present preliminary results show that mHLA-DR is an independent predictor of mortality in septic shock patients. Being a marker of immune failure, low mHLA-DR may provide a rationale for initiating therapy to reverse immunosuppression. After validation of the current results in multicenter studies, mHLA-DR may help to stratify patients when designing a mediator-directed therapy in a time-dependent manner.

Both percentage of gammadelta T lymphocytes and CD3 expression are reduced during septic shock.

OBJECTIVE: The mechanisms involved during sepsis-induced immunosuppression are far from being extensively established. The objective of the present study was to investigate whether two characteristics of T cells were altered in this situation: the percentage of circulating gammadelta T lymphocytes and the level of CD3 expression on T lymphocytes. DESIGN: Observational study. SETTING: Adult intensive care units in a university hospital. PATIENTS: Patients with septic shock (n = 21) and healthy individuals (n = 21). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In patients, we first observed the decreased percentage of gammadelta T lymphocytes in peripheral blood (1% [0.7-3.1], median [interquartile range]) in comparison with healthy individuals (3.5% [2.1-4.8]). Regarding CD3, we measured a highly significant decrease of its expression on both alphabeta and gammadelta T lymphocytes from patients (p < .005), whereas the CD3 mean fluorescence intensities ratio (gammadelta/alphabeta) was not affected: 2.2 [2.1-2.4] and 2.1 [1.9-2.3] in healthy individuals and septic patients, respectively. The magnitude in the decrease of CD3 expression was thus similar in alphabeta and gammadelta cells, suggesting a common down-regulation mechanism for both T-cell lineages. CONCLUSIONS: Combined with a reduced percentage of monocytes expressing human leukocyte antigen-DR, a reduced CD3 expression may be involved in the failure of antigen presentation depicted after septic shock, whereas the diminished percentage of circulating gammadelta T cells could be partly responsible for the elevated incidence of secondary infections. These two observations constitute additional pieces of the complex puzzle of sepsis-induced immunosuppression.

The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives.

The diagnosis of immediate allergy is mainly based upon an evocative clinical history, positive skin tests (gold standard) and, if available, detection of specific IgE. In some complicated cases, functional in vitro tests are necessary. The general concept of those tests is to mimic in vitro the contact between allergens and circulating basophils. The first approach to basophil functional responses was the histamine release test but this has remained controversial due to insufficient sensitivity and specificity. During recent years an increasing number of studies have demonstrated that flow cytometry is a reliable tool for monitoring basophil activation upon allergen challenge by detecting surface expression of degranulation/activation markers (CD63 or CD203c). This article reviews the recent improvements to the basophil activation test made possible by flow cytometry, focusing on the use of anti-CRTH2/DP2 antibodies for basophil recognition. On the basis of a new triple staining protocol, the basophil activation test has become a standardized tool for in vitro diagnosis of immediate allergy. It is also suitable for pharmacological studies on non-purified human basophils. Multicenter studies are now required for its clinical assessment in large patient populations and to define the cut-off values for clinical decision-making.

FcgammaRIIIb and complement component C7 codeficiency in a patient with recurrence of fulminant meningococcal septic shock.

Individuals with deficiencies of the late components of complement exhibit a susceptibility to the recurrence of meningococcal disease with a usually mild clinical presentation. We report the recurrence of fulminant meningococcal disease in a complement component C7-deficient patient. We found a total deficiency of FcgammaRIIIb on neutrophils, which could partially explain the unusually severe clinical presentation.

The Th2 response as monitored by CRTH2 or CCR3 expression is severely decreased during septic shock.

The shift of T lymphocytes toward a Th2 profile during septic shock has been established on the basis of in vitro cytokine production. In the present study, the Th2 response was investigated at the level of cell surface marker expression (whole blood flow cytometry). In 58 patients with septic shock, we observed a reduced CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) expression on Th2 lymphocytes and regulatory T cells in comparison with 39 healthy volunteers. Eosinophils, which constitutively express CRTH2 in healthy individuals, also exhibited low levels of CRTH2 in patients. In addition, eosinophil CCR3 expression (eotaxin receptor, type 2 chemokine) was strongly correlated with CRTH2, suggesting thus an extended modulation of Th2 related molecules. Importantly, the persistence over time of low levels of CRTH2 or CCR3 expression was found in nonsurvivors. We hypothesize that the restoration of CRTH2/CCR3 expression may be an indicator for optimal recovery after septic shock.

The anti-inflammatory response dominates after septic shock: association of low monocyte HLA-DR expression and high interleukin-10 concentration.

The diminished expression of HLA-DR on monocytes has been proposed as a reliable marker of immunosuppression occuring during septic shock. The objective of the present observational study was to establish the time-dependent relation between plasma cytokines interleukin (IL)-10, transforming growth factor (TGF)-beta1, tumor necrosis factor (TNF)-alpha and monocyte HLA-DR expression in 38 adult patients with septic shock. All patients (mortality at 28 days: 42%, mean admission SAPS II score: 54) had decreased HLA-DR expression. This expression was significantly lower in non-survivors at all time points. All patients had elevated IL-10 concentrations, the highest values were found in non-survivors. IL-10 was the sole cytokine to significantly correlate with HLA-DR expression (r: -0.6, p<0.001). TNF and TGF values did not provide any prognostic information. TGF levels from septic patients were even found to be decreased in comparison with normal values which suggested that IL-10 is likely more important than TGF regarding the immunosuppressive properties of septic patients' plasma. This preliminary work showed that, at the systemic level, the anti-inflammatory response dominated after septic shock. Monocyte HLA-DR expression and IL-10 measurement deserve to be determined in parallel in a larger longitudinal study. They might constitute helpful indicators for staging patients and making a decision about whether to institute a therapy with molecules able of reversing sepsis-induced immunosuppression.

Multicenter evaluation of a new immunoassay for procalcitonin measurement on the Kryptor System.

We compared the manually performed LUMItest procalcitonin (PCT) assay with the newly developed fully mechanized Kryptor PCT assay and determined the essential assay characteristics of this assay. The new Kryptor PCT assay was evaluated according to modified NCCLS EP-10/EP-6 protocols in five different laboratories. Samples from 696 patients were assayed using the original LUMItest PCT assay and the new Kryptor PCT assay. Possible interference by hemoglobin, triglycerides and bilirubin was evaluated by spiking patient plasma with the appropriate substances. The functional assay sensitivity (FAS) was determined by analyzing samples with low PCT concentrations. The FAS of the new Kryptor PCT assay was 0.04 ng/ml and the imprecision within- and between-series below 5% and below 10%, respectively. Within the smallest range of determination, from 0.3 ng/ml to 50 ng/ml, common to the LUMItest PCT assay (x) and the Kryptor PCT assay (y) the values correlated well: y=0.64+0.94x, s.xy=2.78 ng/ml. The performance characteristics of the Kryptor PCT assay are fully compatible with the intended clinical use. The assay allows determination of PCT in a turnaround time (TAT) of about 20 minutes and thus is adequate for STAT analyses.

Increased percentage of CD4+CD25+ regulatory T cells during septic shock is due to the decrease of CD4+CD25- lymphocytes.

OBJECTIVE: The elevation of the percentage of regulatory CD4+CD25+ T lymphocytes (Treg) has been recently described during septic shock. The objective of the present study was to investigate whether this increased percentage was due to Treg proliferation. DESIGN: Observational study. SETTING: Adult intensive care units in a university hospital. SUBJECTS: Patients with septic shock (n = 54) and healthy individuals (n = 30). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In patients, we first confirmed the increased percentage of Treg among CD4+ lymphocytes in comparison with healthy individuals. Surprisingly, regarding absolute counting, we demonstrated that both T CD4+ lineages (CD25+ and CD25-) were diminished immediately after the onset of shock. Then, whereas Treg returned rapidly to healthy donors values, CD4+CD25- T lymphocytes remained dramatically reduced. Finally, Foxp3 (Treg-related gene) messenger RNA quantification enabled us to definitively rule out a lack of proliferation since it was not increased during shock. CONCLUSION: The increased percentage of Treg after shock is due not to their proliferation but to a decrease in CD4+CD25- T lymphocyte number. We hypothesize that it might be due to a resistance of Treg to apoptosis processes occurring during septic shock.

Marked elevation of human circulating CD4+CD25+ regulatory T cells in sepsis-induced immunoparalysis.

OBJECTIVE: Immunoparalysis has recently emerged as a possible cause explaining the failure of clinical trials in septic shock. Because human peripheral blood CD4+CD25+ T cells have been characterized as suppressor T cells, we hypothesized they might be increased in sepsis-induced immunoparalysis. DESIGN: Prospective, observational, clinical study. SETTING: Adult intensive care units in a university hospital. SUBJECTS: Patients with septic shock (n = 16) and healthy individuals (n = 36). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In patients with septic shock (mortality rate at 28 days, 56%; mean admission Simplified Acute Physiology Score II, 47), we first illustrated immunoparalysis by showing a severe diminished monocytic human leukocyte antigen (HLA)-DR expression. Afterward, compared with control values, we found in these patients a marked elevation of circulating CD4+CD25+ T cells that were also CD45RO+ and CD69- and overexpressed CTLA-4. Importantly, nonsurvivors (n = 9) presented prolonged lower monocytic HLA-DR expression and higher percentage of CD4+CD25+ T-suppressor T cells. CONCLUSIONS: These data support the concept that the persistence of a pronounced immunoparalysis after septic shock is associated with a poor outcome. Whether CD4+CD25+ T cells directly participate in sepsis-induced immunoparalysis remains to be investigated.

Calcitonin gene related peptide and N-procalcitonin modulate CD11b upregulation in lipopolysaccharide activated monocytes and neutrophils.

OBJECTIVE: Circulating levels of calcitonin gene related peptide (CGRP) and calcitonin precursors, including procalcitonin (PCT) and its free aminopeptide N-procalcitonin (N-PCT), have been found dramatically increased in septic patients. PCT is known to attenuate the chemotaxis of monocytes in response to chemoattractants. This study examined whether CGRP and N-PCT modulate the LPS-induced expression of CD11b, which is one of the major integrins involved in monocyte and neutrophil chemotaxis during a response to microbial infections. DESIGN AND SETTING: In vitro cell culture study in the immunology laboratory of a university hospital. PARTICIPANTS: Healthy volunteers. MEASUREMENTS AND RESULTS: We assessed the effects of N-PCT and CGRP on CD11b expression on monocytes and neutrophils after LPS (2 ng/ml) or fMLP (10(-8) M) challenges. We used a human whole blood model, and measurements were made by flow cytometry. Both peptides in a dose-dependent manner decreased the LPS- and fMLP-induced rise in CD11b in monocytes and neutrophils. As these peptides are thought to act by raising cAMP, we also mimicked their effects with the use of rolipram and forskolin and found similar results. CONCLUSIONS: These findings are in line with recent studies demonstrating anti-inflammatory properties for this family of peptides. CGRP and calcitonin precursors may function as factors suppressing the propagation of inflammation through the inhibition of several processes involved during a response to a bacterial stimulus.

Monitoring of basophil activation using CD63 and CCR3 in allergy to muscle relaxant drugs.

Allergic or pseudoallergic reactions that occur during anesthesia have been increasing for the last few years. To date, the diagnosis of allergy to muscle relaxants remains difficult. In this respect, we developed a flow cytometric method for the study of drug-induced basophil degranulation using CD63 and CCR3. Fifty patients who developed clinical features evocative of allergic reactions immediately after induction of anesthesia were included and classified into two groups. Group 1 (n = 39) comprised true allergic patients, who developed typical signs of shock associated to positive skin testing. Group 2 (n = 11) consisted of patients whose clinical history was not typical and skin testing was negative or nonconclusive. Seventeen control subjects were also studied in this report. We compared data from flow cytometry to skin tests, specific IgE, and histamine release results. Flow cytometry showed a sensitivity of 54%, while that of specific IgE was similar, at 62%. Interestingly, when considering the sensitivity of IgE + CD63 for diagnosis, we reached a sensitivity value of 80%. Of 15 negative results for specific IgE, we found 7 positive CD63 tests, while histamine release gave positive results in only 2 cases. Furthermore, the CD63 protocol showed good specificity (100%). We conclude that our flow cytometry protocol is a promising tool in allergy diagnosis since it is specific and complementary to specific IgE detection.