Cell-mediated immune response: a clinical review of the therapeutic potential of human papillomavirus vaccination.

OBJECTIVE: This clinical review aims to assess the efficacy of human papillomavirus 16/18 (HPV16/18) vaccination on the cell-mediated immune response in women with existing cervical intraepithelial neoplasia or cervical cancer induced by HPV16 or HPV18. DATA SOURCES AND STUDY SELECTION: A focused and thorough literature search conducted in five different databases found 996 publications. Six relevant articles were chosen for further review. In total, 154 patients (>18 years of age) were enrolled in prospective study trials with 3-15 months of follow up. The vaccine applications were administered two to four times. The vaccines contained different combinations of HPV16 and HPV18 and early proteins, E6 and E7. The primary outcome was the cell-mediated immune response. Correlation to clinical outcome (histopathology) and human leukocyte antigen genes were secondary endpoints. RESULTS: All vaccines triggered a detectable cell-mediated immune response, some of which were statistically significant. Correlations between immunological response and clinical outcome (histopathology) were not significant, so neoplasms may not be susceptible to vaccine-generated cytotoxic T cells (CD8(+)). CONCLUSIONS: Prophylactic HPV vaccines have been introduced to reduce the incidence of cervical cancer in young women. Women already infected with HPV could benefit from a therapeutic HPV vaccination. Hence, it is important to continue the development of therapeutic HPV vaccines to lower the rate of HPV-associated malignancies and crucial to evaluate vaccine efficacy clinically. This clinical review represents an attempt to elucidate the theories supporting the development of an HPV vaccine with a therapeutic effect on human papillomavirus-induced malignancies of the cervix.

Immune-mediated thrombotic thrombocytopenic purpura in a Jehovah's Witness - Effectiveness of incorporating extracorporeal immunoadsorption to caplacizumab, steroids and rituximab.

We report the case of a Jehovah's Witness adolescent patient with immune-mediated thrombotic thrombocytopenic purpura after SARS-Cov2 infection successfully treated without therapeutic plasma exchange (TPE) using caplacizumab, corticosteroids, rituximab, and extracorporeal immunoadsorption (EIA). Further patients for whom TPE is not an option might benefit from this approach.

Efficacy and safety of multimodal treatment in nocturnal enuresis - A retrospective cohort study.

INTRODUCTION AND AIM OF THE STUDY: Most treatments of nocturnal enuresis (NE) are targeting the main pathophysiological mechanisms, i.e., excess nocturnal urine production, bladder reservoir dysfunction and inability to awaken to a full bladder. Although many children can be effectively treated with only one treatment modality, there is a significant number of treatment-refractory cases. We experience an increasing tendency to combine treatment modalities in those children. However, there is limited evidence regarding the efficacy and safety of such strategies. MATERIALS AND METHODS: We reviewed files from all NE children seen in our outpatient incontinence clinic between January 1st and December 31st 2017 and identified children refractory to first line treatment receiving a combination of at least two treatment modalities concurrently. Age, gender, wet nights per week before treatment, follow-up time, previous treatment with desmopressin or alarm, phenotype of NE, number of simultaneous treatments tried and response as well as registered side effects during treatment was noted. We registered the outcomes and safety of the treatment modalities and evaluated prognostic factors. RESULTS: We identified 59 children (13 girls) aged 6-15 yrs (mean 9.6 yrs) of whom 30 were monosymptomatic NE (MNE) and 29 were non-monosymptomatic NE (NMNE) patients. They all suffered at least three wet nights per week before treatment. In total, 38 children (61%) became dry on multimodal therapy. Eighteen children (30%) became dry on a combination of two treatment modalities, 16 (27%) on three modalities, and two (3%) on four modalities. Nine children (15%) achieved partial response whereas three (5%) showed no response despite multiple tries with combination therapies. A total of 18 children (30%) reported side effects to one or more of the modalities tried. Side effects that led to discontinuation of the treatment were uncommon (three patients). CONCLUSIONS: Treatment refractory NE represents a challenge for the clinician. Although it seems possible to adequately treat refractory NE patients with multimodal treatment one should be aware of side effects as well as inform the families of the challenges in the treatment of refractory enuresis patients. Future RCT's should focus on providing further evidence for the role of multimodal therapy in NE treatment.

Transcriptomic changes arising during light-induced sporulation in Physarum polycephalum.

BACKGROUND: Physarum polycephalum is a free-living amoebozoan protist displaying a complex life cycle, including alternation between single- and multinucleate stages through sporulation, a simple form of cell differentiation. Sporulation in Physarum can be experimentally induced by several external factors, and Physarum displays many biochemical features typical for metazoan cells, including metazoan-type signaling pathways, which makes this organism a model to study cell cycle, cell differentiation and cellular reprogramming. RESULTS: In order to identify the genes associated to the light-induced sporulation in Physarum, especially those related to signal transduction, we isolated RNA before and after photoinduction from sporulation- competent cells, and used these RNAs to synthesize cDNAs, which were then analyzed using the 454 sequencing technology. We obtained 16,669 cDNAs that were annotated at every computational level. 13,169 transcripts included hit count data, from which 2,772 displayed significant differential expression (upregulated: 1,623; downregulated: 1,149). Transcripts with valid annotations and significant differential expression were later integrated into putative networks using interaction information from orthologs. CONCLUSIONS: Gene ontology analysis suggested that most significantly downregulated genes are linked to DNA repair, cell division, inhibition of cell migration, and calcium release, while highly upregulated genes were involved in cell death, cell polarization, maintenance of integrity, and differentiation. In addition, cell death- associated transcripts were overrepresented between the upregulated transcripts. These changes are associated to a network of actin-binding proteins encoded by genes that are differentially regulated before and after light induction.

Quantitative automated microscopy (QuAM) elucidates growth factor specific signalling in pain sensitization.

BACKGROUND: Dorsal root ganglia (DRG)-neurons are commonly characterized immunocytochemically. Cells are mostly grouped by the experimenter's eye as "marker-positive" and "marker-negative" according to their immunofluorescence intensity. Classification criteria remain largely undefined. Overcoming this shortfall, we established a quantitative automated microscopy (QuAM) for a defined and multiparametric analysis of adherent heterogeneous primary neurons on a single cell base.The growth factors NGF, GDNF and EGF activate the MAP-kinase Erk1/2 via receptor tyrosine kinase signalling. NGF and GDNF are established factors in regeneration and sensitization of nociceptive neurons. If also the tissue regenerating growth factor, EGF, influences nociceptors is so far unknown. We asked, if EGF can act on nociceptors, and if QuAM can elucidate differences between NGF, GDNF and EGF induced Erk1/2 activation kinetics. Finally, we evaluated, if the investigation of one signalling component allows prediction of the behavioral response to a reagent not tested on nociceptors such as EGF. RESULTS: We established a software-based neuron identification, described quantitatively DRG-neuron heterogeneity and correlated measured sample sizes and corresponding assay sensitivity. Analysing more than 70,000 individual neurons we defined neuronal subgroups based on differential Erk1/2 activation status in sensory neurons. Baseline activity levels varied strongly already in untreated neurons. NGF and GDNF subgroup responsiveness correlated with their subgroup specificity on IB4(+)- and IB4(-)-neurons, respectively. We confirmed expression of EGF-receptors in all sensory neurons. EGF treatment induced STAT3 translocation into the nucleus. Nevertheless, we could not detect any EGF induced Erk1/2 phosphorylation. Accordingly, intradermal injection of EGF resulted in a fundamentally different outcome than NGF/GDNF. EGF did not induce mechanical hyperalgesia, but blocked PGE2-induced sensitization. CONCLUSIONS: QuAM is a suitable if not necessary tool to analyze activation of endogenous signalling in heterogeneous cultures. NGF, GDNF and EGF stimulation of DRG-neurons shows differential Erk1/2 activation responses and a corresponding differential behavioral phenotype. Thus, in addition to expression-markers also signalling-activity can be taken for functional subgroup differentiation and as predictor of behavioral outcome. The anti-nociceptive function of EGF is an intriguing result in the context of tissue damage but also for understanding pain resulting from EGF-receptor block during cancer therapy.

Infection prevention and control in patients with cystic fibrosis (CF): Results from a survey in 35 German CF treatment centers.

This survey evaluates whether the Cystic Fibrosis (CF)-specific infection prevention and control (IPC) recommendations released by the Commission for Hospital Hygiene and Infection Prevention (KRINKO) in 2012 have been implemented in specialized German CF facilities. Of 35 participating centers (response rate 32.7%), 37% care for more than 100 patients and 44% treat mainly adults. Clinics for adult CF patients report a shortage of qualified personnel for intensified environmental cleaning. Some hospitals struggle to provide single patient rooms with an adjacent sanitary area to segregate CF patients strictly. Most centers offer at least one decolonization cycle (including systemic and inhalative antibiotics) to patients colonized with MRSA. In CF centers in Germany, the KRINKO IPC recommendations are considered helpful by the attending physicians and thoroughly implemented. There is room for improvement concerning strict segregation of inpatients with CF in single patient rooms, in particular in large CF centers mainly caring for adults.

A first glimpse at the transcriptome of Physarum polycephalum.

BACKGROUND: Physarum polycephalum, an acellular plasmodial species belongs to the amoebozoa, a major branch in eukaryote evolution. Its complex life cycle and rich cell biology is reflected in more than 2500 publications on various aspects of its biochemistry, developmental biology, cytoskeleton, and cell motility. It now can be genetically manipulated, opening up the possibility of targeted functional analysis in this organism. METHODS: Here we describe a large fraction of the transcribed genes by sequencing a cDNA library from the plasmodial stage of the developmental cycle. RESULTS: In addition to the genes for the basic metabolism we found an unexpected large number of genes involved in sophisticated signaling networks and identified potential receptors for environmental signals such as light. In accordance with the various developmental options of the plasmodial cell we found that many P. polycephalum genes are alternatively spliced. Using 30 donor and 30 acceptor sites we determined the splicing signatures of this species. Comparisons to various other organisms including Dictyostelium, the closest relative, revealed that roughly half of the transcribed genes have no detectable counterpart, thus potentially defining species specific adaptations. On the other hand, we found highly conserved proteins, which are maintained in the metazoan lineage, but absent in D. discoideum or plants. These genes arose possibly in the last common ancestor of Amoebozoa and Metazoa but were lost in D. discoideum. CONCLUSION: This work provides an analysis of up to half of the protein coding genes of Physarum polycephalum. The definition of splice motifs together with the description of alternatively spliced genes will provide a valuable resource for the ongoing genome project.

Antimicrobial efficacy and compatibility of solid copper alloys with chemical disinfectants.

INTRODUCTION: Chemical disinfection is state of the art in preventing spread of infectious agents in the healthcare setting. Additionally, the antimicrobial properties of solid copper alloy surfaces against various microorganisms have recently been substantiated. Thus, antimicrobially active copper surfaces may serve as an additional barrier against distribution of pathogenic microorganisms and be combined with chemical disinfection measures in the hospital. The aim of this study was therefore to investigate on a quantitative basis whether the combination of chemical disinfectants with copper alloy surfaces results in an overall compromised, combined or even synergistic antimicrobial efficacy. METHODS: Experiments were carried out using the quantitative carrier test devised by the German Society for Hygiene and Microbiology (DGHM) to study antimicrobial efficacy of chemical disinfectants. Requirements for microbicidal efficacy as defined by prEN 14885 were applied. The chemical disinfectants tested in our study contained alcohols (ethanol, 1-propanol), quaternary ammonium compounds (benzalkonium chloride) and glutaraldehyde as actives. Quantitative carrier tests were carried out on different carriers (tiles, copper alloy discs, stainless steel discs) using Pseudomonas aeruginosa, Staphylococcus aureus, Kocuria rhizophila and Candida albicans as test organisms. RESULTS: For the alcohol-based disinfectant no difference in antimicrobial efficacy was observed when applied to antimicrobial active copper alloy carriers, tiles or stainless steel discs. For all test organisms microbial contamination was reduced to the detection limit of < 1 log (CFU/ml) within a contact time of 2 min indicating a ≥ 5 log reduction for the tested bacteria and a ≥ 4 log reduction for the yeast, as being requested for chemical disinfectants by prEN 14885. In order to elucidate a potential synergism the chemical disinfectant based on quaternary ammonium compounds (benzalkonium chloride) and glutaraldehyde was used at a sub-effective concentration. Hence, no complete reduction of microbial contamination was achieved on stainless steel or tile carriers for Pseudomonas aeruginosa and Candida albicans. Interestingly, when using copper alloy carriers complete reduction indicating a ≥ 5 log reduction for P. aeruginosa and a ≥ 4 log reduction for C. albicans was detected. Thus, data of this study indicates that solid copper alloy surfaces and disinfectants synergize. CONCLUSIONS: According to this data, commercially available disinfectants based on alcohol, quaternary ammonium compounds and aldehyde can effectively be combined in a dual strategy with solid copper alloy surfaces to reduce microbial contamination.

Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization.

Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a K(D) in the millimolar range, dimeric 3C had a significantly increased binding affinity with K(D) values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.

Clinical impact of two different intraoperative parathyroid hormone assays in primary and renal hyperparathyroidism.

BACKGROUND: Intraoperative parathyroid hormone (PTH) monitoring predicts successful surgery for primary hyperparathyroidism (pHPT). In renal HPT, intraoperative PTH assays can define whether parathyroid resection is adequate. METHODS: Intraoperative PTH was measured with two different immunometric assays (Immulite Turbo DPC and ADVIA Centaur assay) in 91 patients undergoing parathyroidectomy for primary (n=57) and renal (n=34) hyperparathyroidism. PTH was monitored preoperatively, 10, 20, and 30 min after parathyroidectomy and 24 h postoperatively. RESULTS: Ten minutes after parathyroidectomy, intraoperative PTH dropped into the normal range (<7.6 pmol/l) in 84% of patients with pHPT and tertiary HPT as measured with the ADVIA Centaur assay (PTH-A), compared with 100% of the samples measured with the Immulite Turbo DPC assay (PTH-I; P=0.0082). Twenty minutes after parathyroidectomy for secondary HPT, intraoperative PTH decreased to the normal range in 100% measured with PTH-I compared with 50% measured with PTH-A (P=0.009). Then, 24 h postoperatively, PTH-I and PTH-A levels were within the normal range in all of the successfully treated patients. Both assays correctly identified six patients with persistent disease and another patient with a double adenoma in pHPT. CONCLUSIONS: In patients undergoing parathyroidectomy for primary or renal HPT, PTH levels decreasing to the normal range indicated successful surgery in all of the patients as measured with the PTH-I assay. Comparing the two assays, PTH-I was able to quantify the intraoperative PTH decay more quickly than PTH-A.

HLA type-independent generation of antigen-specific T cells for adoptive immunotherapy.

Adoptive immunotherapy with antigen-specific T cells has been successfully used to treat certain infectious diseases and cancers. Although more patients may profit from T cell therapy, its more frequent use is restricted by limitations in current T cell generation strategies. The most commonly applied peptide-based approaches rely on the knowledge of relevant epitopes. Therefore, T cells cannot be generated for diseases with unknown epitopes or for patients with unfavorable HLA types. We developed a peptide-based approach for HLA type-independent generation of specific T cells against various proteins. It is based on short-time stimulation with peptide libraries that cover most CD4(+) and CD8(+) T cell epitopes of given proteins. The procedure requires no prior knowledge of epitopes because libraries are synthesized solely on the basis of the protein's amino acid sequence. Stimulation is followed by immunomagnetic selection of activated IFN-gamma-secreting cells and nonspecific expansion. To evaluate the protocol, we generated autologous T cells specific for a well-characterized antigen, the human cytomegalovirus phosphoprotein 65 (pp65). Generated T cell lines consisted of pp65-specific CD4(+) and CD8(+) lymphocytes that displayed antigen-specific killing and proliferation. The protocol combines the biosafety of peptide-based approaches with HLA type independence and may help to advance adoptive immunotherapy in the future.

Identifying site-specific substitution rates.

A maximum likelihood framework for estimating site-specific substitution rates is presented that does not require any prior assumptions about the rate distribution. We show that, when the branching pattern of the underlying tree is known, the analysis of pairs of positions is sufficient to estimate site-specific rates. In the abscense of a known topology, we introduce an iterative procedure to estimate simultaneously the branching pattern, the branch lengths, and site-specific substitution rates. Simulations show that the evolutionary rate of fast-evolving sites can be reliably inferred and that the accuracy of rate estimates depends mainly on the number of sequences in the data set. Thus, large sets of aligned sequences are necessary for reliable site-specific rate estimates. The method is applied to the complete mitochondrial DNA sequence of 53 humans, providing a complete picture of the site-specific substitution rates in human mitochondrial DNA.

Ancient DNA analyses reveal high mitochondrial DNA sequence diversity and parallel morphological evolution of late pleistocene cave bears.

Cave bears (Ursus spelaeus) existed in Europe and western Asia until the end of the last glaciation some 10,000 years ago. To investigate the genetic diversity, population history, and relationship among different cave bear populations, we have determined mitochondrial DNA sequences from 12 cave bears that range in age from about 26,500 to at least 49,000 years and originate from nine caves. The samples include one individual from the type specimen population, as well as two small-sized high-Alpine bears. The results show that about 49,000 years ago, the mtDNA diversity among cave bears was about 1.8-fold lower than the current species-wide diversity of brown bears (Ursus arctos). However, the current brown bear mtDNA gene pool consists of three clades, and cave bear mtDNA diversity is similar to the diversity observed within each of these clades. The results also show that geographically separated populations of the high-Alpine cave bear form were polyphyletic with respect to their mtDNA. This suggests that small size may have been an ancestral trait in cave bears and that large size evolved at least twice independently.