Occurrence and trends of Clostridioides difficile infections in hospitalized patients: a prospective multi-centre cohort study in six German university hospitals, 2016-2020.

BACKGROUND: For Clostridioides difficile infections (CDIs) in Germany no longitudinal multi-centre studies with standardized protocols for diagnosing CDI are available. Recent evaluations of general surveillance databases in Germany indicate a downward trend in CDI rates. We aimed to describe the actual burden and trends of CDI in German university hospitals from 2016 to 2020. METHODS: Our study was a prospective multi-centre study covering six German university hospitals. We report the data in total, stratified by year, by medical specialty as well as by CDI severity. Multi-variable regression analyses were performed to assess risk factors for severe CDI. RESULTS: We registered 3780 CDI cases among 1,436,352 patients. The median length of stay (LOS) of CDI cases was 20 days (interquartile range 11-37) compared with a general LOS of 4.2 days. In-hospital all-cause mortality in CDI patients was 11.7% (N = 444/3780), while mortality attributed to CDI was 0.4% (N = 16/3761). CDI recurrence rate was comparatively low at 7.2%. The incidence density of severe healthcare-associated healthcare onset (HAHO)-CDI showed a significant decrease from 2.25/10,000 patient days (pd) in 2016 to 1.49/10,000 pd in 2020 (trend calculation P=0.032). CONCLUSIONS: Compared with a European point-prevalence study in 2013/2014, where overall CDI incidence density was 11.2 cases/10,000 pd in Germany (EUCLID), we see in our study halved overall CDI rates of 5.6 cases/10,000 pd in 2020. Our study shows current data on the distribution of CDI cases in German university hospitals and thus provides international comparative data on the key indicators of CDI.

Gene expression profiling meta-analysis reveals novel gene signatures and pathways shared between tuberculosis and rheumatoid arthritis.

Tuberculosis (TB) is among the leading causes of death by infectious diseases. An epidemiological association between Mycobacterium tuberculosis infection and autoimmune diseases like rheumatoid arthritis (RA) has been reported but it remains unclear if there is a causal relationship, and if so, which molecular pathways and regulatory mechanisms contribute to it. Here we used a computational biology approach by global gene expression meta-analysis to identify candidate genes and pathways that may link TB and RA. Data were collected from public expression databases such as NCBI GEO. Studies were selected that analyzed mRNA-expression in whole blood or blood cell populations in human case control studies at comparable conditions. Six TB and RA datasets (41 active TB patients, 33 RA patients, and 67 healthy controls) were included in the downstream analysis. This approach allowed the identification of deregulated genes that had not been identified in the single analysis of TB or RA patients and that were co-regulated in TB and RA patients compared to healthy subjects. The genes encoding TLR5, TNFSF10/TRAIL, PPP1R16B/TIMAP, SIAH1, PIK3IP1, and IL17RA were among the genes that were most significantly deregulated in TB and RA. Pathway enrichment analysis revealed 'T cell receptor signaling pathway', 'Toll-like receptor signaling pathway,' and 'virus defense related pathways' among the pathways most strongly associated with both diseases. The identification of a common gene signature and pathways substantiates the observation of an epidemiological association of TB and RA and provides clues on the mechanistic basis of this association. Newly identified genes may be a basis for future functional and epidemiological studies.

Frequent loss of expression of the pro-apoptotic protein Bim in renal cell carcinoma: evidence for contribution to apoptosis resistance.

Renal cell carcinoma (RCC) is resistant to chemotherapy, and this resistance is mirrored by a high apoptosis resistance of many RCC lines in vitro. Here, we report the loss of the pro-apoptotic BH3-only protein Bim in a large part of clinical RCC cases and provide evidence for a functional relevance of this loss. Immunohistochemistry of clear cell renal cell carcinoma cases and corresponding normal kidney showed strong Bim reactivity in renal tubules of all cases but loss of Bim in 35 of 45 RCC samples. Out of nine RCC cell lines investigated, six showed strongly diminished or undetectable levels of Bim protein by western blotting. Four RCC lines of varying apoptosis sensitivity were analysed further. Bcl-2, Bcl-x(L), Mcl-1, Bax and Bak expression did not correlate with apoptosis sensitivity. All cell lines underwent apoptosis upon forced expression of Bax and Bim, suggesting an upstream difference. In all four lines, adriamycin induced p53 but not its targets Puma or Noxa. However, apoptosis sensitivity correlated with levels of Bim protein. Bim siRNA reduced apoptosis sensitivity in a susceptible cell line. Furthermore, inhibition of histone deacetylation restored Bim expression in cell lines. These data suggest that Bim has a function as a tumor suppressor in RCC.

Modified vaccinia virus Ankara protein F1L is a novel BH3-domain-binding protein and acts together with the early viral protein E3L to block virus-associated apoptosis.

Infection with viruses often protects the infected cell against external stimuli to apoptosis. Here we explore the balance of apoptosis induction and inhibition for infection with the modified vaccinia virus Ankara (MVA), using two MVA mutants with experimentally introduced deletions. Deletion of the E3L-gene from MVA transformed the virus from an inhibitor to an inducer of apoptosis. Noxa-deficient mouse embryonic fibroblasts (MEF) were resistant to MVA-DeltaE3L-induced apoptosis. When the gene encoding F1L was deleted from MVA, apoptosis resulted that required Bak or Bax. MVA-DeltaF1L-induced apoptosis was blocked by Bcl-2. When expressed in HeLa cells, F1L blocked apoptosis induced by forced expression of the BH3-only proteins, Bim, Puma and Noxa. Finally, biosensor analysis confirmed direct binding of F1L to BH3 domains. These data describe a molecular framework of how a cell responds to MVA infection by undergoing apoptosis, and how the virus blocks apoptosis by interfering with critical steps of its signal transduction.

Apoptosis. A sticky business.

Many proteins that resemble Bcl-2 or bind to it have been found using techniques that reflect interactions in vitro or depend on DNA homology, but we still do not know how this master regulator of apoptosis works.

Requirements for proteolysis during apoptosis.

The key effector proteins of apoptosis are a family of cysteine proteases termed caspases. Following activation of caspases, biochemical events occur that lead to DNA degradation and the characteristic morphological changes associated with apoptosis. Here we show that cytoplasmic extracts activated in vitro by proteinase K were able to cleave the caspase substrate DEVD-7-amino-4-methylcoumarin, while neither proteinase K nor nonactivated extracts were able to do so alone. Caspase-like activity was inhibited by the specific caspase inhibitor DEVD-aldehyde and by the protease inhibitor iodoacetamide, but not by N-ethylmaleimide. When added to isolated nuclei, the activated extracts caused internucleosomal DNA degradation and morphological changes typical of apoptosis. As DNA cleavage and morphological changes could be inhibited by N-ethylmaleimide but not by iodoacetamide, we conclude that during apoptosis, caspase activation causes activation of another cytoplasmic enzyme that can be inhibited by N-ethylmaleimide. Activity of this enzyme is necessary for activation of endonucleases, DNA cleavage, and changes in nuclear morphology.

Uncouplers of oxidative phosphorylation can enhance a Fas death signal.

Recent work suggests a participation of mitochondria in apoptotic cell death. This role includes the release of apoptogenic molecules into the cytosol preceding or after a loss of mitochondrial membrane potential DeltaPsim. The two uncouplers of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 2, 4-dinitrophenol (DNP) reduce DeltaPsim by direct attack of the proton gradient across the inner mitochondrial membrane. Here we show that both compounds enhance the apoptosis-inducing capacity of Fas/APO-1/CD95 signaling in Jurkat and CEM cells without causing apoptotic changes on their own account. This amplification occurred upstream or at the level of caspases and was not inhibited by Bcl-2. The effect could be blocked by the cowpox protein CrmA and is thus likely to require caspase 8 activity. Apoptosis induction by staurosporine in Jurkat cells as well as by Fas in SKW6 cells was unaffected by CCCP and DNP. The role of cytochrome c during Fas-DNP signaling was investigated. No early cytochrome c release from mitochondria was detected by Western blotting. Functional assays with cytoplasmic preparations from Fas-DNP-treated cells also indicated that there was no major contribution by cytochrome c or caspase 9 to the activation of effector caspases. Furthermore, an increase of rhodamine-123 uptake into intact cells, which has been explained by mitochondrial swelling, occurred considerably later than the caspase activation and was blocked by Z-VAD-fmk. These data show that uncouplers of oxidative phosphorylation can presensitize some but not all cells for a Fas death signal and provide information about the existence of separate pathways in the induction of apoptosis.

Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9.

We have generated rat monoclonal antibodies that specifically recognise caspase-2 from many species, including mouse, rat and humans. Using these antibodies, we have investigated caspase-2 expression, subcellular localisation and processing. We demonstrate that caspase-2 is expressed in most tissues and cell types. Cell fractionation and immunohistochemistry experiments show that caspase-2 is found in the nuclear and cytosolic fractions, including a significant portion present in the Golgi complex. We found that caspase-2 is processed in response to many apoptotic stimuli but experiments with caspase-2 deficient mice demonstrated that it is not required for apoptosis of thymocytes or dorsal root ganglia (DRG) neurons in response to a variety of cytotoxic stimuli. Caspase-2 processing does not occur in thymocytes lacking Apaf-1 or caspase-9, suggesting that in this cell type, activation of caspase-2 occurs downstream of apoptosome formation.

In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins.

Mitochondrial apoptosis is controlled by proteins of the B-cell lymphoma 2 (Bcl-2) family. Pro-apoptotic members of this family, known as BH3-only proteins, initiate activation of the effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), which is counteracted by anti-apoptotic family members. How the interactions of Bcl-2 proteins regulate cell death is still not entirely clear. Here, we show that in the absence of extrinsic apoptotic stimuli Bak activates without detectable contribution from BH3-only proteins, and cell survival depends on anti-apoptotic Bcl-2 molecules. All anti-apoptotic Bcl-2 proteins were targeted via RNA interference alone or in combinations of two in primary human fibroblasts. Simultaneous targeting of B-cell lymphoma-extra large and myeloid cell leukemia sequence 1 led to apoptosis in several cell types. Apoptosis depended on Bak whereas Bax was dispensable. Activator BH3-only proteins were not required for apoptosis induction as apoptosis was unaltered in the absence of all BH3-only proteins known to activate Bax or Bak directly, Bcl-2-interacting mediator of cell death, BH3-interacting domain death agonist and p53-upregulated modulator of apoptosis. These findings argue for auto-activation of Bak in the absence of anti-apoptotic Bcl-2 proteins and provide evidence of profound differences in the activation of Bax and Bak.

Proteolytic specificity of caspases is required to signal the appearance of apoptotic morphology.

The caspase family of cysteine proteases is essential for implementation of physiological cell death. Since a wide variety of cellular proteins is cleaved by caspases during apoptosis, it has been predicted that digestion of proteins crucial to maintaining the life of a cell is central to apoptosis. To assess the role of the proteolytic destruction during apoptosis, we introduced the non-specific protease proteinase K into intact cells. This introduction led to extensive digestion of cellular proteins, including physiological caspase-substrates. Caspase-3-like activity was induced rapidly, followed by morphological signs of apoptosis such as membrane blebbing and nuclear condensation. The caspase inhibitor Z-VAD-fmk inhibited the appearance of these morphological changes without reducing the extent of intracellular proteolysis by proteinase K. Loss of integrity of the cell membrane, however, was not blocked by Z-VAD-fmk. This system thus generated conditions of extensive destruction of caspase substrates by proteinase K in the absence of apoptotic morphology. Taken together, these experiments suggest that caspases implement cell death not by protein destruction but by proteolytic activation of specific downstream effector molecules.

Localization and molecular forms of galanin in human adrenals: elevated levels in pheochromocytomas.

Galanin immunoreactivity was measured by RIA, using antibodies directed against both the non-C- and C-terminal positions of porcine galanin, in tissue extracts of normal adrenals and pheochromocytomas and also in the plasma of normal subjects and patients with pheochromocytomas. No C-terminal galanin-like immunoreactivity was detected in plasma or tissue, suggesting differences in the amino acid sequence of human compared with porcine galanin. A non-C-terminally directed antibody was, therefore, used to characterize human galanin immunoreactivity by gel permeation chromatography and reverse phase high pressure liquid chromatography and to localize it by immunocytochemistry. The galanin content of whole adrenal gland was 2.6 +/- 0.9 (+/- SEM) pmol/g (n = 5). In contrast, however, pheochromocytomas had much greater concentrations (21 +/- 2.3 pmol/g; n = 16). Gel chromatography and reverse phase high pressure liquid chromatography revealed 2 molecular forms of galanin immunoreactivity with identical elution positions in both normal adrenals and tumors. The concentration of galanin in plasma from both normal subjects and pheochromocytoma patients was below the detection limit of the assay (less than 10 pmol/liter). Using immunocytochemistry, galanin was localized to scattered cells or clusters of tumor cells in 5 of 11 pheochromocytomas and only a few chromaffin cells and cortical nerve fibers in normal adrenals.

Detection of caspase-activation in intact lymphoid cells using standard caspase substrates and inhibitors.

Members of the caspase family of proteases are important in the implementation of apoptotic cell death. These caspases are intracellularly activated upon a death stimulus, and exhibit a distinctive proteolytic activity which transmits a death signal and readily detected by measuring the cleavage of synthetic substrates in cell extracts. In this report, we show that apoptosis-associated caspase activation can be recorded not only in cell lysates but also in intact lymphoid cells with commercially available peptides which are either biotinylated or carry an amino-methylcoumarin (AMC) group. Incubation of intact cells induced to undergo apoptosis with Ac-Asp-Glu-Val-Asp-AMC (DEVD-AMC) leads to the release of AMC in amounts very similar to the amounts released when cell extracts are prepared and incubated with DEVD-AMC. This release can be detected by a fluorescence read-out and is blocked by caspase-inhibitors such as Ac-DEVD-cho or Z-VAD-fmk. Similarly, labelling of intact cells with the biotinylated peptides Tyr-Val-Ala-Asp-cmk (YVAD-cmk) or YVAD-faom permits the detection of active caspases by affinity blotting and the detection of apoptotic cells by FACS analysis. These methods enable the investigator to detect at the single-cell level those cells which have activated their caspases and to evaluate such activation without the need for lysis of the cells.

Inhibition of etoposide-induced apoptotic events by azide.

Azide is reported to be an inducer of necrotic rather than apoptotic cell death. Using various parameters of cell death, we demonstrate here that azide is capable of completely inhibiting apoptosis induced by VP-16/etoposide. Azide was found systematically to protect Jurkat cells against VP-16-induced effects as determined by an array of biochemical and morphological parameters. The cells were able partially to recover proliferative activity following removal of the drugs. We conclude that azide inhibits apoptosis induced by VP-16 at an early point in the apoptotic pathway and that inhibition of apoptosis might be the mechanism of necrosis-induction by azide.

Inhibition of nuclear factor kappa B and induction of apoptosis in T-lymphocytes by sulfasalazine.

1. Chronic inflammatory diseases have been shown to be associated with NF-kappaB activation and impaired apoptosis of immune cells. The aim of the present study was to investigate if sulfasalazine and its colonic metabolites 5-aminosalicylic acid (5ASA) and sulfapyridine affect NF-kappaB/Rel activation and viability of T-lymphocytes. 2. Sulfasalazine inhibits NF-kappaB/Rel activation in the murine T-lymphocyte cell line RBL5 using electrophoretic mobility shift assays. In transfection assays sulfasalazine treatment for 4 h inhibits kappaB-dependent transcription with an IC50 value of approximately 0.625 mM. 3. Higher doses or prolonged treatment result in cell death of T-lymphocytes in a dose- and time-dependent manner. Cell death is caused by apoptosis as judged by DNA fragmentation, annexin V and Apo 2.7 staining. Induction of apoptosis is a fast event with 50% apoptotic cells after a 4 h incubation with 2.5 mM sulfasalazine. The ED50 value for apoptosis induction after 24 h treatment was approximately 0.625 mM. 4. In contrast, 5ASA and sulfapyridine neither inhibit NF-kappaB/Rel activation nor induce apoptosis in T-lymphocytes at doses up to 5.0 mM. 5. These results demonstrate that sulfasalazine, but not 5ASA or sulfapyridine, strongly inhibits NF-kappaB activation and potently induces apoptosis in T-lymphocytes. Inhibition of NF-kappaB/Rel activation and subsequent clearance of activated T-lymphocytes by apoptosis might thus explain the beneficial effects of sulfasalazine in the treatment of chronic inflammatory disorders.

Tumor progression, histologic grading and DNA-ploidy as predictive factors of lymphogenous metastasis in primary carcinoma of the Fallopian tube.

UNLABELLED: The bad prognosis of primary carcinoma of the Fallopian tube (FTC), with 5-year overall survival rates of only 35%, is particularly ascribed to lymphogenous metastasis. Yet, we know very little on the pathophysiologic factors on which this lymphogenous metastasis is based. The present study, therefore, aims at evaluating the influence of intra-abdominal tumor progression and tumor-cell anaplasia on lymphogenous metastasis in FTC. We studied 41 cases of FTC, who had been subjected to radical lymphadenectomy during primary operation in a retrospective analysis. Staging was done by International Federation of Gynecology and Obstetrics-classification. Histologic grading and nuclear DNA-content (DNA-index) were used for evaluating tumor-cell anaplasia. Histologic grading discriminated between highly differentiated (G1), moderately dedifferentiated (G2), and dedifferentiated (G3) tumors. According to their DNA-indices, tumors were separated into three groups: DNA-index < or =1.1 (euploid cases), DNA-indices between 1.1 and 2.0 (cases of intermediate ploidy), and DNA-index >2.0 (aneuploid cases). The overall incidence of lymph node metastases was 43.9%. There was no correlation between histologic grading and DNA-index (P=0.98). Lymphogenous metastasis set in after the tumor had transgressed the tube (intra-abdominal stage II). Further intra-abdominal tumor progression (including omentum, liver, or peritoneum) significantly increases the incidence of lymph node metastases (P=0.02). There was only a single G1-tumor that had already disseminated into the lymph, all other cases of lymph node metastases were found in G2- or G3-tumors. DNA-index and the extent of lymphogenous metastases were not found to be correlated (P=0.74). CONCLUSIONS: The extent of lymphogenous metastases in FTC depends above all on intra-abdominal tumor progression. This fact has clinical consequences as the indication for lymphadenectomy can be obtained directly during operation. The results of histologic grading are of no impact on the surgical proceedings; the determination of DNA-ploidy is negligible.

Prognostic impact of DNA content and AUER classification in primary fallopian tube carcinoma.

DNA ploidy has been studied in 61 primary fallopian tube carcinomas using image-cytometry. The investigation also included survival analysis, and ploidy classification according to AUER was performed in order to evaluate its prognostic impact for fallopian tube carcinoma. A high number of aneuploid cases were observed (79% aneuploid vs. 21% euploid tumors). The high incidence of aneuploid tumors was consistently observed among all FIGO-stages as well as all groups of histologic grading. There was no correlation between ploidy and FIGO-stage or histologic grading. Patients with euploid DNA content showed a median survival of 34 months compared to 24 months for aneuploid cases (log-rank, P = 0.83). No correlation between the AUER classification and FIGO-stage or histologic grading could be observed. Tumors with an AUER type I and II (75th quantile 41 months) showed a better outcome than tumors with AUER III and IV (75th quantile 19 months). Although these results did not reach statistical significance (P = 0.07), a trend could be observed. Therefore AUER classification may be useful as an objective prognostic parameter. The high incidence of aneuploid tumors could be an expression of the high biologic aggressiveness of primary fallopian tube cancer which has been repeatedly mentioned in the past.

Localization and distribution of vasoactive neuropeptides in the human placenta.

Neuropeptides play an important role in the regional regulation of blood flow and hormone secretion. Few studies report the presence of peptides in the human placenta. Our experiment evaluates neuropeptides in the human placenta using immunocytochemical techniques. Representative tissue sections from full-term placentae were fixed immediately after delivery and processed into paraffin sections or frozen. They were treated with multiple immunofluorescence, streptavidin-biotin-peroxidase complex and immunogold-silver staining techniques in combination with well-established monoclonal and polyclonal antibodies, using appropriate absorption controls to ensure the validity of the staining. Vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), neuropeptide tyrosine (NPY), galanin, somatostatin, met-enkephaline, helodermin and substance P-like immunoreactivities were demonstrated within decidual cells. Endothelin-1 was found in both trophoblasts and endothelial cells. Peptide immunoreactivities in the human placenta especially at the decidual interface between mother and fetus supports a role for the diffuse neuroendocrine system (DNES) in the regulation of placental blood flow critical for fetal growth and development.

Cloning of mouse RP-8 cDNA and its expression during apoptosis of lymphoid and myeloid cells.

A partial-length cDNA encoding a cell death-associated gene designated RP-8 was cloned from rat thymocytes by Owens and co-workers (Mol. Cell. Biol. 11, 4177-4188, 1991). They reported that transcription of RP-8 was induced when rat thymocytes were caused to undergo apoptosis triggered by either radiation or treatment with dexamethasone. To study the role of RP-8 in cell death in the mouse, we have cloned a full-length mouse RP-8 cDNA from a mouse thymus cDNA library. The mouse RP-8 gene encodes of protein of 343 amino acids with 92% amino acid identity to the rat RP-8 protein. Expression of mouse RP-8 was not altered in a factor-dependent myeloid line induced to undergo apoptosis by growth factor withdrawal, or when a lymphoid cell line was triggered to undergo apoptosis by irradiation. Although these data do not prove that RP-8 is not a cell death gene, they show that RP-8 expression is not sufficient for apoptosis, and that transcriptional up regulation of RP-8 is not universally associated with apoptosis.