Markus Hufnagel

ORAI1-mediated calcium influx is required for human cytotoxic lymphocyte degranulation and target cell lysis

Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca2+ entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8+ effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1β and the cytokines TNF-α and IFN-γ on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca2+ influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition.

Subtle differences in CTL cytotoxicity determine susceptibility to hemophagocytic lymphohistiocytosis in mice and humans with Chediak-Higashi syndrome.

Perforin-mediated cytotoxicity is important for controlling viral infections, but also for limiting immune reactions. Failure of this cytotoxic pathway leads to hemophagocytic lymphohistiocytosis (HLH), a life-threatening disorder of uncontrolled T-cell and macrophage activation. We studied susceptibility to HLH in 2 mouse strains (souris and beige(J)) and a cohort of patients with partial defects in perforin secretion resulting from different mutations in the LYST gene. Although both strains lacked NK-cell cytotoxicity, only souris mice developed all clinical and histopathologic signs of HLH after infection with lymphocytic choriomeningitis virus. The 2 strains showed subtle differences in CTL cytotoxicity in vitro that had a large impact on virus control in vivo. Whereas beige(J) CTLs eliminated lymphocytic choriomeningitis virus infection, souris CTLs failed to control the virus, which was associated with the development of HLH. In LYST-mutant patients with Chediak-Higashi syndrome, CTL cytotoxicity was reduced in patients with early-onset HLH, whereas it was retained in patients who later or never developed HLH. Thus, the risk of HLH development is set by a threshold that is determined by subtle differences in CTL cytotoxicity. Differences in the cytotoxic capacity of CTLs may be predictive for the risk of Chediak-Higashi syndrome patients to develop HLH.

Serological and Genetic Diversity of Capsular Polysaccharides in Enterococcus faecalis

ABSTRACT Enterococci possess capsular carbohydrate antigens that are targets of opsonic antibodies. These antigens may be used to develop alternative options for the treatment and prevention of enterococcal infections. The present study was done to analyze the diversity of capsular polysaccharides in Enterococcus faecalis. Four type-specific sera were used in an enzyme-linked immunosorbent assay format to detect polysaccharide antigen extracted from bacterial cell walls. A total of 55% of a collection of 29 E. faecalis strains could be grouped into one of four serogroups. Additional analysis of the strains by opsonophagocytic assays revealed agreement between the results of the two methods for 72% of the isolates. An additional four strains could be assigned to a serogroup on the basis of opsonic killing by sera with antibodies against the four prototypes strains, provisionally named CPS-A to CPS-D. The results of the two methods disagreed for one strain (4%). When the results of both methods were combined, 66% of the strains could be classified. One strain had to be assigned to two serogroups. The assignments to the four serogroups were confirmed by analysis of the genetic organization of the biosynthetic capsular polysaccharide (cps) locus. All strains grouped into serotypes CPS-A and CPS-B possessed only the cpsA and cpsB genes, while all strains grouped into serogroups CPS-C and CPS-D possessed an additional eight or nine genes. Our results suggest the existence of a limited number of E. faecalis capsule serotypes, and we provisionally propose four serotypes, named CPS-A to CPS-D, and the respective prototype strains for these families.

New LightCycler PCR for Rapid and Sensitive Quantification of Parvovirus B19 DNA Guides Therapeutic Decision-Making in Relapsing Infections

ABSTRACT Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically ≥5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus.

International External Quality Assurance for Laboratory Identification and Typing of Streptococcus agalactiae (Group B Streptococci)

ABSTRACT We report the results from the first international multicenter external quality assessment (EQA) studies for molecular and serological typing of group B streptococcus (GBS) strains as part of DEVANI (Design of a Vaccine against Neonatal Infections), a pan-European program. A questionnaire-based surveillance was undertaken among eight laboratories participating in DEVANI and six laboratories not participating in DEVANI from 13 countries in order to assess their current microbiological procedures for GBS screening, diagnosis, and typing. GBS strains from three EQA distributions were characterized using molecular and serological methods based on GBS capsular polysaccharide typing. Participants were asked to test the first distribution using their current serotyping and genotyping methods. The Strep-B-Latex agglutination method was the most widely used method, with a typeability value of >90%. A multiplex PCR assay for GBS capsular gene typing was also used by 2 of 14 centers, which achieved a typeability value of 93%; this assay detected only 9 of 10 GBS capsular polysaccharide genes. From the second and third EQA studies, standardized protocols were prepared for serological and molecular typing of GBS strains based on the Strep-B-Latex agglutination method and a novel multiplex PCR assay that detected all 10 GBS capsular types (Ia to IX). These standardized protocols are being used by many European laboratories, and as the use of these methods increases, it is imperative to continuously improve and assess laboratory performance and offer training to any laboratories that have technical difficulties.

Japanese Encephalitis: Defining Risk Incidence for Travelers to Endemic Countries and Vaccine Prescribing From the UK and Switzerland

BACKGROUND Large numbers of Western travelers visit countries endemic for Japanese encephalitis (JE). The risk of infection is unknown. This study attempts at estimating a risk incidence for visitors from two European countries with the available data. METHODS Using the total number of case reports between 1978 and 2008, the number of visits made by European tourists to endemic regions, and total doses of vaccines sold in the two study countries, the risk incidence of JE in travelers was estimated. The proportion of vaccinated visitors to endemic regions was retrieved from the data of two travel clinics (in London and Basel) and related to vaccine prescribing in UK and Swiss travelers. RESULTS In 2004, an estimated 0.16% to 0.3% of UK and Swiss travelers were vaccinated against JE, with no surveillance reports of JE cases. Between 116,000 and 152,000 European travelers would receive vaccination. More than 99% travel to endemic countries without vaccination. Only 40 cases of JE infection have been reported in travelers for the past 30 years. The risk incidence is thus 1.3 per year in 7.1 million visits of the 17 million European travelers who are at a potential risk of JE infection. CONCLUSIONS This study and the analysis of the existing literature support the recommendation that all travelers should be informed about the risk of JE infection but also suggest that there is no evidence for justifying a general recommendation for JE vaccination in travelers to endemic areas.

Enterococcal colonization of infants in a neonatal intensive care unit: associated predictors, risk factors and seasonal patterns

BackgroundDuring and shortly after birth, newborn infants are colonized with enterococci. This study analyzes predictors for early enterococcal colonization of infants in a neonatal intensive care unit and describes risk factors associated with multidrugresistant enterococci colonization and its seasonal patterns.MethodsOver a 12-month period, we performed a prospective epidemiological study in 274 infants admitted to a neonatal intensive care unit. On the first day of life, we compared infants with enterococcal isolates detected in meconium or body cultures to those without. We then tested the association of enterococcal colonization with peripartal predictors/risk factors by using bivariate and multivariate statistical methods.ResultsTwenty-three percent of the infants were colonized with enterococci. The three most common enterococcal species were E. faecium (48% of isolates), E. casseliflavus (25%) and E. faecalis (13%). Fifty-seven percent of the enterococci found were resistant to three of five antibiotic classes, but no vancomycin-resistant isolates were observed. During winter/spring months, the number of enterococci and multidrug-resistant enterococci were higher than in summer/fall months (p = 0.002 and p < 0.0001, respectively). With respect to enterococcal colonization on the first day of life, predictors were prematurity (p = 0.043) and low birth weight (p = 0.011). With respect to colonization with multidrug-resistant enterococci, risk factors were prematurity (p = 0.0006), low birth weight (p < 0.0001) and prepartal antibiotic treatment (p = 0.019). Using logistic regression, we determined that gestational age was the only parameter significantly correlated with multidrug-resistant enterococci colonization. No infection with enterococci or multidrugresistant enterococci in the infants was detected. The outcome of infants with and without enterococcal colonization was the same with respect to death, necrotizing enterocolitis, intracerebral hemorrhage and bronchopulmonary dysplasia.ConclusionIn neonatal intensive care units, an infants susceptibility to early colonization with enterococci in general, and his or her risk for colonization with multidrug-resistant enterococci in particular, is increased in preterm newborns, especially during the winter/spring months. The prepartal use of antibiotics with no known activity against enterococci appears to increase the risk for colonization with multidrug-resistant enterococci.

Genetic differences between invasive and noninvasive neonatal group B streptococcal isolates.

Background: Streptococcus agalactiae, also known as group B streptococcus (GBS), is the most common cause of neonatal sepsis and meningitis. To improve our understanding of the pathogenesis of neonatal GBS sepsis, better knowledge of clonal relatedness and diversity among invasive and noninvasive GBS isolates is critical. Methods: In a Germany-based study, invasive neonatal GBS isolates were compared with noninvasive isolates from neonates in whom sepsis was suspected, but whose blood cultures were sterile. The comparison was conducted by means of pulsed-field gel electrophoresis and surface protein gene profiling. In addition, multilocus sequence typing was performed on invasive and noninvasive isolates of the most frequent invasive serotype III. Results: Pulsed-field gel electrophoresis analysis of noninvasive GBS showed a remarkably more diverse fingerprinting pattern than that of invasive isolates. In contrast to invasive strains, noninvasive isolates did not show any clustering. Surface protein gene profiling also showed significantly different distribution patterns between the 2 panels of isolates. Multilocus sequence typing of invasive and noninvasive serotype III isolates revealed the same clonal complexes, but displayed different sequence types (ST); ST-17 was most common (68.6%) among invasive strains, whereas ST-389 (clonal complex-19) was predominant among noninvasive strains (47.8%). Conclusions: Our results illustrate a large molecular diversity among neonatal noninvasive GBS strains. Invasive strains, however, represent only a small proportion of the noninvasive GBS population. These findings suggest a selection process that prefers more virulent strains during invasion.

Significant decline in the erythromycin resistance of group A streptococcus isolates at a German paediatric tertiary care centre

Group A streptococcus (GAS) is considered to be a major pathogen of bacterial tonsillopharyngitis in children. Although GAS is generally susceptible to penicillin, macrolides are often used as the second-line treatment. Over the last several decades, the rising macrolide resistance of GAS has been detected in several countries. With the current study, we aimed to determine the development of macrolide resistance at our paediatric centre. From March 2006 to May 2009, 350 GAS isolates were tested for susceptibility to erythromycin, azithromycin, clindamycin, penicillin and cefotaxime. Macrolide-resistant isolates were screened for the presence of genes related to macrolide resistance (mefA, ermB, ermTR, prtF1). In comparison to a prior study at our hospital, the erythromycin resistance rate decreased significantly from 13.6 to 2.6%. This effect may be attributable to a more restrictive use of macrolides in children in our region.

Persistence of human bocavirus DNA in immunocompromised children.

Human bocavirus is frequently detected in immunocompetent as well as in immunocompromised children. However, the course of infection in immunocompromised children is still poorly investigated. In the present study, we describe 4 cases of repeat human bocavirus detection in the presence of severe immunodeficiency. In the view of homologous viral sequences identified in serial samples, possible persistence and reactivation in these patients are discussed.