Monocytes show immunoregulatory capacity on CD4+ T cells in a human in-vitro model of extracorporeal photopheresis.

Extracorporeal photopheresis (ECP) is a widely used immunomodulatory therapy for the treatment of various T cell-mediated disorders such as cutaneous T cell lymphoma (CTCL), graft-versus-host disease (GvHD) or systemic sclerosis. Although clinical benefits of ECP are already well described, the underlying mechanism of action of ECP is not yet fully understood. Knowledge on the fate of CD14+ monocytes in the context of ECP is particularly limited and controversial. Here, we investigated the immunoregulatory function of ECP treated monocytes on T cells in an in-vitro ECP model. We show that ECP-treated monocytes significantly induce proinflammatory T cell types in co-cultured T cells, while anti-inflammatory T cells remain unaffected. Furthermore, we found significantly reduced proliferation rates of T cells after co-culture with ECP-treated monocytes. Both changes in interleukin secretion and proliferation were dependent on cell-contact between monocytes and T cells. Interestingly, blocking interactions of programmed death ligand 1 (PD-L1) to programmed death 1 (PD-1) in the in-vitro model led to a significant recovery of T cell proliferation. These results set the base for further studies on the mechanism of ECP, especially the regulatory role of ECP-treated monocytes.

Granulocytic myeloid-derived suppressor cells (GR-MDSC) accumulate in cord blood of preterm infants and remain elevated during the neonatal period.

Preterm delivery is the leading cause of perinatal morbidity and mortality. Among the most important complications in preterm infants are peri- or postnatal infections. Myeloid-derived suppressor cells (MDSC) are myeloid cells with suppressive activity on other immune cells. Emerging evidence suggests that granulocytic MDSC (GR-MDSC) play a pivotal role in mediating maternal-fetal tolerance. The role of MDSC for postnatal immune-regulation in neonates is incompletely understood. Until the present time, nothing was known about expression of MDSC in preterm infants. In the present pilot study, we quantified GR-MDSC counts in cord blood and peripheral blood of preterm infants born between 23 + 0 and 36 + 6 weeks of gestation (WOG) during the first 3 months of life and analysed the effect of perinatal infections. We show that GR-MDSC are increased in cord blood independent of gestational age and remain elevated in peripheral blood of preterm infants during the neonatal period. After day 28 they drop to nearly adult levels. In case of perinatal or postnatal infection, GR-MDSC accumulate further and correlate with inflammatory markers C-reactive protein (CRP) and white blood cell counts (WBC). Our results point towards a role of GR-MDSC for immune-regulation in preterm infants and render them as a potential target for cell-based therapy of infections in these patients.

Regulatory T cell frequencies are increased in preterm infants with clinical early-onset sepsis.

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (Tregs ) are potential candidates for the ontogenetic control of immune activation and tissue damage in preterm infants. It was the aim of our study to characterize lymphocyte subsets and in particular CD4(+) CD25(+) forkhead box protein 3 (FoxP3)(+) Tregs in peripheral blood of well-phenotyped preterm infants (n = 117; 23 + 0 - 36 + 6 weeks of gestational age) in the first 3 days of life in comparison to term infants and adults. We demonstrated a negative correlation of Treg frequencies and gestational age. Tregs were increased in blood samples of preterm infants compared to term infants and adults. Notably, we found an increased Treg frequency in preterm infants with clinical early-onset sepsis while cause of preterm delivery, e.g. chorioamnionitis, did not affect Treg frequencies. Our data suggest that Tregs apparently play an important role in maintaining maternal-fetal tolerance, which turns into an increased sepsis risk after preterm delivery. Functional analyses are needed in order to elucidate whether Tregs have potential as future target for diagnostics and therapeutics.

Clinical and laboratory characteristics of paediatric and adolescent index cases with venous thromboembolism and antithrombin deficiency. An observational multicentre cohort study.

Venous thromboembolism [TE] is a multifactorial disease and antithrombin deficiency [ATD] constitutes a major risk factor. In the present study the prevalence of ATD and the clinical presentation at TE onset in a cohort of paediatric index cases are reported. In 319 unselected paediatric patients (0.1-18 years) from 313 families, recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. 21 of 319 paediatric patients (6.6%), corresponding to 16 of 313 families (5.1%), were AT-deficient with confirmed underlying AT gene mutations. Mean age at first TE onset was 14 years (range 0.1 to 17). Thrombotic locations were renal veins (n=2), cerebral veins (n=5), deep veins (DVT) of the leg (n=9), DVT & pulmonary embolism (n=4) and pelvic veins (n=1). ATD co-occurred with the factor-V-Leiden mutation in one and the prothrombin G20210A mutation in two children. In 57.2% of patients a concomitant risk factor for TE was identified, whereas 42.8% of patients developed TE spontaneously. A second TE event within primarily healthy siblings occurred in three of 313 families and a third event among siblings was observed in one family. In an unselected cohort of paediatric patients with symptomatic TE, the prevalence of ATD adjusted for family status was 5.1%. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high risk population.

Neutrophilic myeloid-derived suppressor cells in cord blood modulate innate and adaptive immune responses.

Neonates show an impaired anti-microbial host defence, but the underlying immune mechanisms are not understood fully. Myeloid-derived suppressor cells (MDSCs) represent an innate immune cell subset characterized by their capacity to suppress T cell immunity. In this study we demonstrate that a distinct MDSC subset with a neutrophilic/granulocytic phenotype (Gr-MDSCs) is highly increased in cord blood compared to peripheral blood of children and adults. Functionally, cord blood isolated Gr-MDSCs suppressed T cell proliferation efficiently as well as T helper type 1 (Th1), Th2 and Th17 cytokine secretion. Beyond T cells, cord blood Gr-MDSCs controlled natural killer (NK) cell cytotoxicity in a cell contact-dependent manner. These studies establish neutrophilic Gr-MDSCs as a novel immunosuppressive cell subset that controls innate (NK) and adaptive (T cell) immune responses in neonates. Increased MDSC activity in cord blood might serve as key fetomaternal immunosuppressive mechanism impairing neonatal host defence. Gr-MDSCs in cord blood might therefore represent a therapeutic target in neonatal infections.

Defective plasmacytoid dendritic cell-NK cell cross-talk in HIV infection.

HIV viremia is associated with a wide range of immune dysfunctions that contribute to the immunocompromised state. HIV viremia has been shown to have a broad effect on several immune cell types and/or their interactions that are vital for mounting an effective immune response. In this study, we investigated the integrity of plasmacytoid dendritic cell (pDC)-NK cell interactions among HIV viremic, aviremic, and seronegative individuals. We describe a critical defect in the ability of pDCs from HIV-infected individuals to secrete IFN-alpha and TNF and subsequently activate NK cells. We also describe an inherent defect on NK cells from HIV-infected individuals to respond to pDC-secreted cytokines. Furthermore, we were able to demonstrate a direct effect of HIV trimeric gp120 on NK cells in vitro similar to that described ex vivo. Finally, we were able to establish that the HIV gp120-mediated suppressive effect on NK cells was a result of its binding to the integrin alpha(4)beta(7) expressed on NK cells. These findings suggest a novel mechanism by which HIV is capable of suppressing an innate immune function in infected individuals.

[Prenatal diagnosis of an inguino-scrotal hernia in a fetus with trisomy 18]. / Pränatale Diagnose einer Skrotalhernie bei einem Feten mit Trisomie 18.

In spite of its prevalence in neonates, an inguino-scrotal hernia is a rarely detected condition in prenatal life. The sonographic findings, i. e., a scrotal mass with intestinal peristalsis, the differential diagnosis, and possible associations between a scrotal hernia and chromosomal aberrations are described by presenting the case of a fetus with trisomy 18 and major structural anomalies.

A predicted operon map for Mycobacterium tuberculosis.

The prediction of operons in Mycobacterium tuberculosis (MTB) is a first step toward understanding the regulatory network of this pathogen. Here we apply a statistical model using logistic regression to predict operons in MTB. As predictors, our model incorporates intergenic distance and the correlation of gene expression calculated for adjacent gene pairs from over 474 microarray experiments with MTB RNA. We validate our findings with known examples from the literature and experimentation. From this model, we rank each potential operon pair by the strength of evidence for cotranscription, choose a classification threshold with a true positive rate of over 90% at a false positive rate of 9.1%, and use it to construct an operon map for the MTB genome.

Invasion and persistent intracellular colonization of erythrocytes. A unique parasitic strategy of the emerging pathogen Bartonella.

The expanding genus Bartonella includes zoonotic and human-specific pathogens that can cause a wide range of clinical manifestations. A productive infection allowing bacterial transmission by blood-sucking arthropods is marked by an intraerythrocytic bacteremia that occurs exclusively in specific human or animal reservoir hosts. Incidental human infection by animal-adapted bartonellae can cause disease without evidence for erythrocyte parasitism. A better understanding of the intraerythrocytic lifestyle of bartonellae may permit the design of strategies to control the reservoir and transmittable stages of these emerging pathogens. We have dissected the process of Bartonella erythrocyte parasitism in experimentally infected animals using a novel approach for tracking blood infections based on flow cytometric quantification of green fluorescent protein-expressing bacteria during their interaction with in vivo-biotinylated erythrocytes. Bacteremia onset occurs several days after inoculation by a synchronous wave of bacterial invasion into mature erythrocytes. Intracellular bacteria replicate until reaching a stagnant number, which is sustained for the remaining life span of the infected erythrocyte. The initial wave of erythrocyte infection is followed by reinfection waves occurring at intervals of several days. Our findings unravel a unique bacterial persistence strategy adapted to a nonhemolytic intracellular colonization of erythrocytes that preserves the pathogen for efficient transmission by blood-sucking arthropods.

STRAP: editor for STRuctural Alignments of Proteins.

STRAP is a comfortable and extensible tool for the generation and refinement of multiple alignments of protein sequences. Various sequence ordered input file formats are supported. These are the SwissProt-,GenBank-, EMBL-, DSSP- PDB-, MSF-, and plain ASCII text format. The special feature of STRAP is the simple visualization of spatial distances C(alpha)-atoms within the alignment. Thus structural information can easily be incorporated into the sequence alignment and can guide the alignment process in cases of low sequence similarities. Further STRAP is able to manage huge alignments comprising a lot of sequences. The protein viewers and modeling programs INSIGHT, RASMOL and WEBMOL are embedded into STRAP. STRAP is written in JAVA: The well-documented source code can be adapted easily to special requirements. STRAP may become the basis for complex alignment tools in the future.

Conservation of substructures in proteins: interfaces of secondary structural elements in proteasomal subunits.

It is observed that during divergent evolution of two proteins with a common phylogenetic origin, the structural similarity of their backbones is often preserved even when the sequence similarity between them decreases to a virtually undetectable level. Here we analyzed, whether the conservation of structure along evolution involves also the local atomic structures in the interfaces between secondary structural elements. We have used as study case one protein family, the proteasomal subunits, for which 17 crystal structures are known. These include 14 different subunits of Saccharomyces cerevisiae, 2 subunits of Thermoplasma acidophilum and one subunit of Escherichia coli. The structural core of the 17 proteasomal subunits has 23 secondary structural elements. Any two adjacent secondary structural elements form a molecular interface consisting of two molecular patches. We found 61 interfaces that occurred in all 17 subunits. The 3D shape of equivalent molecular patches from different proteasomal subunits were compared by superposition. Our results demonstrate that pairs of equivalent molecular patches show an RMSD which is lower than that of randomly chosen patches from unrelated proteins. This is true even when patch comparisons with identical residues were excluded from the analysis. Furthermore it is known that the sequential dissimilarity is correlated to the RMSD between the backbones of the members of protein families. The question arises whether this is also true for local atomic structures. The results show that the correlation of individual patch RMSD values and local sequence dissimilarities is low and has a wide range from 0 to 0.41, however, it is surprising that there is a good correlation between the average RMSD of all corresponding patches and the global sequence dissimilarity. This average patch RMSD correlates slightly stronger than the C(alpha)-trace RMSD to the global sequence dissimilarity.

Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.

More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.

Bipolar clamping improves the sensitivity of mutation detection by temperature gradient gel electrophoresis.

Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)-product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a "GC-clamp" is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.

A pooling strategy for heterozygote screening of the delta F508 cystic fibrosis mutation.

A theoretical and practical approach to economize the analysis of large DNA sample numbers for identifying heterozygosity of the delta F508 mutation causing cystic fibrosis is presented. Sample pooling can reduce the number of polymerase chain reaction (PCR) tests for this mutation by up to 77%. Based on a mathematical model, the optimal number (n) of samples to be united in one pool is 24 for a German population with a delta F508 heterozygosity incidence of about 1/35. We show that the PCR method is sufficient to detect one heterozygote for the delta F508 mutation in a pool of up to 49 non-delated DNA samples.