Cytomegalovirus infection and rehospitalization rates after allogeneic hematopoietic stem cell and solid organ transplantation: a retrospective cohort study using German claims data.

PURPOSE: This study aimed to describe the cytomegalovirus (CMV) infection rate, rehospitalizations, and comorbidities following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and solid organ transplantation (SOT). METHODS: Patients who received allo-HSCT or SOT in 01/07/2015-30/06/2018 were identified using anonymized German claims data. The transplantation-related hospital admission date was defined as the index date, and patients were followed for up to 12 months (or death, first event relevant). The frequency of CMV infections (confirmed outpatient/inpatient diagnoses, ICD-10-GM codes: B25.-/B27.1) and the rate, number, and duration of all-cause rehospitalizations in the follow-up period were evaluated. RESULTS: A total of 226 allo-HSCT and 250 SOT patients were identified (mean age 52.8 years, 38.9% female). During the 12 months after transplantation, 29.2% of allo-HSCT patients and 16.8% of SOT patients received a CMV diagnosis. The majority of these diagnoses were given during the initial hospitalization or within the following 3 months. Across transplantation types, CMV patients had more hospital readmission days per patient-year (allo-HSCT 93.3 vs. 49.4, p = 0.001; SOT 42.0 vs. 20.7, p = 0.005), with a longer mean duration of readmissions (allo-HSCT 22.4 vs. 15.4 days, p < 0.001; SOT 11.6 vs. 7.5 days, p = 0.003). Comorbidity burden in transplantation patients was substantial, with several diagnoses being significantly more common among patients with CMV vs. non-CMV. One-year mortality did not differ significantly between patients with/without CMV. CONCLUSION: Burden of transplant recipients with CMV in terms of rehospitalizations and comorbidities is substantial, highlighting the need for improved CMV prevention and treatment.

Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion.

Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood-brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na(+),K(+)-ATPase during luminal expansion of brain ventricles. These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis.

Participation of the second extracellular loop of claudin-5 in paracellular tightening against ions, small and large molecules.

Tight junctions control paracellular permeability. Here, we analyzed the impact of residues in the second extracellular loop (ECL2) of mouse claudin-5 on paracellular permeability. Stable expression of claudin-5(wild type) in MDCK-II cells-but not that of mutants R145A, Y148A, Y158A or E159Q-increased transepithelial electrical resistance and decreased fluorescein permeation. Expression of claudin-5(Y148A), (Y158A) or (E159Q) enhanced permeability of FITC-dextran(10 kDa), which was unchanged in cells expressing claudin-5(wild type) or claudin-5(R145A). In contrast, targeting to tight junctions, strand morphology and tight junction assembly were unchanged. It is concluded that R145 is unessential for trans-interaction of claudin-5, but necessary for tightening against small solutes and ions. The highly conserved residues Y148, Y158 and E159 in ECL2 of claudin-5 contribute to homo- and/or heterophilic trans-interaction between classic claudins and thereby tighten the paracellular space against ions, small and large molecules. These results provide novel insights into the molecular function of tight junctions.

Formation of tight junction: determinants of homophilic interaction between classic claudins.

Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation.

Structure and function of extracellular claudin domains.

Most claudins are tight junction (TJ)-forming proteins. However, their interaction on the molecular level remains unresolved. It is hypothesized that the extracellular loops specify these claudin functions. It is assumed that the first extracellular loop (ECL1) is critical for determining the paracellular tightness and the selective paracellular ion permeability, and that the second extracellular loop may cause narrowing of the paracellular cleft. Using a combination of site-directed mutagenesis and homology modeling for the second extracellular loop (ECL2) of claudin-5, we found several amino acids important for claudin folding and/or trans-interaction to claudins in neighboring cells. These sensitive residues are highly conserved within one group of claudins, whereas the corresponding positions in the remaining claudins show a large sequence variety. Further functional data and analysis of sequence similarity for all claudins has led to their differentiation into two groups, designated as classic claudins (1-10, 14, 15, 17, 19) and nonclassic claudins (11-13, 16, 18, 20-24). This also corresponds to conserved structural features at ECL1 for classic claudins. Based on this, we propose a hypothesis for different pore-forming claudins. Pore formation or tightness is supported by the spatial encounter of a surplus of repulsing or attracting amino acid types at ECL1. A pore is likely opened by repulsion of equally charged residues, while an encounter of unequally charged residues leads to tight interaction. These considerations may reveal the ECLs of claudins as decisive submolecular determinants that specify the function of a claudin.

Molecular determinants of the interaction between Clostridium perfringens enterotoxin fragments and claudin-3.

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116-319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10-13, 15, 16, 18-20, and 22. Substitution peptide mapping identified the central motif (148)NPL(150)VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn(148) and Leu(150) of full-length claudin-3 in the binding. CPE-(116-319) and CPE-(194-319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116-319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116-319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116-319) and CPE-(194-319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.