Serum IgM Antibodies Contribute to High Levels of Opsonophagocytic Activities in Toddlers Immunized with a Single Dose of the 9-Valent Pneumococcal Conjugate Vaccine

In immunogenicity trials of pneumococcal conjugate vaccines (PCVs), only IgG antibody concentrations to pneumococcal capsular polysaccharides (PPSs) are usually determined, along with the opsonophagocytic activity (OPA) of antipneumococcal antibodies. We aimed to determine the role of both IgG and IgM in OPA in toddlers receiving one dose of 9-valent PCV (PCV9). The IgG and IgM antibody concentrations to PPSs of serotypes 6A, 9V, 14, 19F, and 23F were measured by enzyme immunoassay in sera from toddlers (ages 18 to 35 months) 1 month after a single PCV9 dose. The OPA for the same serotypes was measured by multiplexed opsonophagocytosis assay (MOPA). Further, IgG and IgM concentrations and MOPA were measured to PPS of serotypes 6A, 14, and 19F in sera collected 12 months after vaccination. The detected MOPA titers were high in comparison to the IgG concentrations 1 month after immunization. The IgM concentrations were higher than IgG concentrations for serotypes 6A and 14 (P < 0.001) and as high as IgG for serotypes 9V, 19F, and 23F. Correlation of the IgM antibody concentrations with MOPA (r = 0.35 to 0.65) was stronger compared to that of the IgG antibodies (r = 0.07 to 0.41). The depletion of IgG antibodies in three sets of pooled sera only slightly decreased the OPA activity against serotype 14. At 12 months after immunization, 50 to 100% of serum samples still showed detectable MOPA activity against serotypes 6A, 14, and 19F. Our results suggest that IgM contributes to OPA 1 month after a single PCV9 vaccination in toddlers and that functionally active IgM and IgG antibodies persist for at least a year.     

Mutations in bone morphogenetic protein receptor 1B cause brachydactyly type A2

Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening and lateral deviation of the index fingers and, to a variable degree, shortening and deviation of the first and second toes. We performed linkage analysis in two unrelated German families and mapped a locus for BD type A2 to 4q21-q25. This interval includes the gene bone morphogenetic protein receptor 1B (BMPR1B), a type I transmembrane serinethreonine kinase. In one family, we identified a T599 --> A mutation changing an isoleucine into a lysine residue (I200K) within the glycine/serine (GS) domain of BMPR1B, a region involved in phosphorylation of the receptor. In the other family we identified a C1456 --> T mutation leading to an arginine-to-tryptophan amino acid change (R486W) in a highly conserved region C-terminal of the BMPR1B kinase domain. An in vitro kinase assay showed that the I200K mutation is kinase-deficient, whereas the R486W mutation has normal kinase activity, indicating a different pathogenic mechanism. Functional analyses with a micromass culture system revealed a strong inhibition of chondrogenesis by both mutant receptors. Overexpression of mutant chBmpR1b in vivo in chick embryos by using a retroviral system resulted either in a BD phenotype with shortening and/or missing phalanges similar to the human phenotype or in severe hypoplasia of the entire limb. These findings imply that both mutations identified in human BMPR1B affect cartilage formation in a dominant-negative manner.     

Efficacy of cognitive behavioral internet-based therapy in parents after the loss of a child during pregnancy: pilot data from a randomized controlled trial

The loss of a child during pregnancy can be a traumatic event associated with long-lasting grief and psychological distress. This study examined the efficacy of an internet-based cognitive behavioral therapy program for mothers after pregnancy loss. In a randomized controlled trial with a waiting list control group, 83 participants who had lost a child during pregnancy were randomly allocated either to 5 weeks of internet therapy or to a 5-week waiting condition. Within a manualized cognitive behavioral treatment program, participants wrote ten essays on loss-specific topics. Posttraumatic stress, grief, and general psychopathology, especially depression, were assessed pretreatment, posttreatment, and at 3-month follow-up. Intention-to-treat analyses and completer analyses were performed. Relative to controls, participants in the treatment group showed significant improvements in posttraumatic stress, grief, depression, and overall mental health, but not in anxiety or somatization. Medium to large effect sizes were observed, and the improvement was maintained at 3-month follow-up. This internet-based cognitive behavioral therapy program represents an effective treatment approach with stable effects for women after pregnancy loss. Implementation of the program can thus help to improve the health care provision for mothers in this traumatic loss situation.     

Regulation of myocardial connexins during hypertrophic remodelling.

Cardiac hypertrophic remodelling, initiated by signalling cascades in response to increased workload, injury or intrinsic disease, is initially adaptive. However, prolonged hypertrophy as a consequence of pathological stress leads to maladaptive changes that increase the risk for fatal ventricular arrhythmias. One of these changes is the remodelling of myocardial gap junctions, which provide for electrical coupling of adjacent cardiomyocytes. Myocardial gap junctions are composed of three connexin isotypes, connexin40 (Cx40), -43 (Cx43), and -45 (Cx45) and each display a characteristic developmental and regional expression pattern. Alterations in the distribution and expression of Cx43, the predominant isoform in the adult ventricles, has been the main focus of examination in humans, experimental animal models and cultured cardiomyocytes in response to hypertrophy. The molecular mechanisms and signalling pathways underlying these changes have been studied less thoroughly. In this review we summarize what is known about the remodelling of myocardial gap junctions during hypertrophy, the putative underlying mechanisms and functional consequences thereof.     

Reliable detection and quantitation of viral nucleic acids in oral fluid: Liquid phase-based sample collection in conjunction with automated and standardized molecular assays

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.     

Dermcidin-Derived Peptides Show a Different Mode of Action than the Cathelicidin LL-37 against Staphylococcus aureus

Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.Antimicrobial peptides (AMPs) serve as a first line of innate host defense in many species such as plants, amphibians, insects, and mammals. AMPs show a broad-spectrum antimicrobial activity against a wide range of pathogens including bacteria, fungi, and enveloped viruses (51). Most gene-encoded AMPs are synthesized as proforms, which are subsequently processed into mature peptides of various lengths. A common feature of most of these peptides is that they are cationic and form amphipathic structures (3). The mode of action of most AMPs is incompletely understood. It is believed that most AMPs kill microorganisms by membrane permeation either via pore formation or via membrane disintegration like that induced by the human cathelicidin LL-37. However, membrane disruption may not reflect the complex processes involved in the killing of microorganisms (5, 48). In addition, several AMPs clearly act differently and intracellular target sites have been identified (15). The mode of action has been unraveled for only a few AMPs which act via defined targets, such as the lantibiotic nisin, which specifically binds to bacterial lipid II, a membrane-bound component involved in peptidoglycan (PG) synthesis (7, 46). Similarly, the lantibiotic mersacidin interferes with transglycosylation and PG synthesis in gram-positive bacteria by direct targeting of lipid II (6). Furthermore, buforin II kills microorganisms by disruption of critical intracellular processes such as the inhibition of macromolecular biosynthesis or by interacting with nucleic acids inside the microorganisms (33). For several AMPs it has been demonstrated that the charge and the composition of the bacterial cell envelope determine sensitivity to AMPs (37). Staphylococcus aureus mutants lacking specific modifications in the bacterial envelope are highly susceptible to a variety of cationic AMPs. For example, incorporation of d-alanine into S. aureus teichoic acids by the dltA enzymes or the lysinylation of phosphatidylglycerol by mprF confers resistance to defensins, protegrins, and other AMPs by repulsion of the cationic peptides (36).Dermcidin (DCD) was identified by our group as a human AMP which is constitutively expressed in eccrine sweat glands and secreted into sweat (40). By postsecretory proteolytic processing in human sweat, the precursor protein gives rise to several truncated DCD peptides varying in length from 25 to 48 amino acids and with net charges between −2 and +2 (2, 10, 38). Several DCD peptides show antimicrobial activity against pathogenic microorganisms such as S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans, Staphylococcus epidermidis, Pseudomonas putida, and methicillin-resistant S. aureus as well as rifampin- and isoniazid-resistant Mycobacterium tuberculosis (9, 25, 40, 41, 45). We were able to show that DCD-derived peptides are also active under high-salt conditions and in a buffer resembling human sweat (40). Antimicrobially active DCD peptides, namely, the anionic peptides DCD-1L (48-mer) and DCD-1 (47-mer) and the cationic peptides SSL-25 (25-mer) and SSL-23 (23-mer), are derived from the C-terminal region of the precursor protein. Interestingly, these peptides have diverse and overlapping spectra of activity which are independent of the net peptide charge (41). In previous studies we showed that DCD peptides interact with the bacterial cell envelope and kill gram-negative bacteria without forming pores in membranes (41). In this study we investigated the mode of antimicrobial activity of DCD-derived peptides in more detail and studied bacterial factors that govern sensitivity or tolerance to DCD in the model microorganism S. aureus. In our first approach, we tried to identify the bacterial surface molecules to which DCD peptides bind. Second, we analyzed the bacterial response to DCD peptide challenge. Finally, we analyzed bacterial mutants to elucidate the mechanisms determining DCD sensitivity.     

Platelet function assessed by shear-induced deposition of split triple-dose apheresis concentrates treated with pathogen reduction technologies

BACKGROUND: Pathogen inactivation/reduction technology (PRT) may alter quality of stored platelet (PLT) concentrates (PCs). Therefore, PLT adhesion and aggregation should be studied before transfusion of PRT-treated PLTs.
STUDY DESIGN AND METHODS: A three-arm in vitro study on triple-dose apheresis PCs (n = 9) was conducted. Split single units were designated to PRT treatment with either a riboflavin (M)- or a psoralen (I)-based technique and compared to untreated controls (C). Samples were taken on Days 0, 1, 5, 7, and 8 to assess PLT function via a cone and plate(let) analyzer, flow cytometric P-selectin expression, and turbidometric aggregation response to thrombin receptor–activating peptide 6 (TRAP-6).
RESULTS: P-selectin expression increased and TRAP-6–inducible expression decreased steadily in all units until reaching a plateau on Day 5 of storage. PRT-treated units demonstrated significant (p ≤ 0.008) differences to C units due to a more pronounced upregulation in P-selectin expression after PRT treatment. The same was true for TRAP-6 after Day 5 of storage. C units were significantly superior over PRT-treated units (p ≤ 0.002), among which M yielded higher values than I (p ≤ 0.008). Although M demonstrated increased shear-induced PLT deposition that remained stable during storage (p = 0.082), surface coverage significantly declined in C (p = 0.047) and especially in I (p = 0.003), but differences between M, C, and I did not reach significance. All units exhibited a slight increase in aggregate size that remained comparable throughout storage (p ≥ 0.141).
CONCLUSIONS: Irrespective of storage-related changes in PLT activation and turbidometric aggregation response, riboflavin-based PRT seemed to benefit shear-induced PLT adhesion. The impact of this finding for PLT function and thrombogenesis in vivo must await clinical evaluation.