A stringent preemptive protocol reduces cytomegalovirus disease in the first 6 months after kidney transplantation.

BACKGROUND: The optimal strategy to prevent cytomegalovirus (CMV) disease after kidney transplantation continues to be open to debate. The preemptive approach requires regular determination of CMV viremia and prompt initiation of therapy. METHODS: We retrospectively compared the incidence of CMV disease during two periods at our center: A first phase (P1, n = 84 kidney recipients), during which time the intensity of surveillance was determined by the responsible physician, was compared to a second phase (P2, n = 74), when a stringent protocol of CMV surveillance was required for all patients. The preemptive approach was applied for all CMV risk groups; prophylaxis was optional in the case of treatment for rejection or delayed graft function in the intermediate- and high-risk group. Follow-up was truncated at 6 months after transplant surgery. CMV syndrome was differentiated from asymptomatic replication by the presence of at least one systemic symptom, while diagnosis of CMV end-organ disease required histological confirmation. RESULTS: Immunosuppression was similar in the two periods. CMV prophylaxis was used equally (26 %) in both periods. The probability for asymptomatic viremia episodes was not different for patients in P1 and P2 regardless of the prevention strategy. For patients following the preemptive strategy, the probability for CMV disease was increased during P1 (p = 0.016), despite fewer PCR assays being performed in phase 2. Protocol violations were only observed during P1. CONCLUSIONS: The probability of CMV disease episodes (CMV syndrome and CMV end-organ disease) was substantially reduced using a very stringent protocol. This study highlights the crucial importance of a stringent protocol with optimal adherence by all caregivers if the preemptive strategy is to be successful.

Basic characteristics and safety of donation in related and unrelated haematopoietic progenitor cell donors - first 10 years of prospective donor follow-up of Swiss donors.

Since July 2007 prospective life-long follow-up (FU) for unrelated (URD) and related donors (RD) is mandatory in Switzerland and data on every allogeneic haematopoietic progenitor cell (HPC) donation are collected prospectively. We report the real-world experience of HPC donation during a 10-year study period (01.07.2007-30.06.2017) with basic characteristics and FU data. 1105 donors underwent 1155 HPC donation procedures. Eighty percent of first donations performed by 802 (73%) RDs and 303 (27%) URDs were peripheral blood stem cells (PBSC), 20% bone marrow (BM). Male donors were over-represented as URD (60% male vs 40% female). Main differences between RDs and URDs concerned age and pre-existing health disorders. RDs were significantly older at first donation (median age 48 years) compared to URD (34 years, p < 0.0001) and had more pre-existing health problems: 25% vs 9% in URD (p < 0.0001). No fatal complications occurred, collection related severe adverse events (SAE) after first donation were not significantly different between groups (RD 1.2%, URD 0.99%), incidence rates for neoplastic and autoimmune diseases did not exceed the rates of the general population. RDs are a more heterogeneous and potentially more vulnerable group, but if donor evaluation is performed appropriately, HPC donation is still safe.

A pilot iron substitution programme in female blood donors with iron deficiency without anaemia.

BACKGROUND AND OBJECTIVES: Blood donation can contribute to iron deficiency. The possibly resulting anaemia importantly affects donor return rate. The determination of serum ferritin levels revealed iron deficiency in many non-anaemic premenopausal female blood donors at our Institution. We started an iron substitution programme targeting this donor group to prevent anaemia and enhance donor retain. MATERIALS AND METHODS: Women aged≤50 with haemoglobin levels adequate for donation and serum ferritin≤10 ng/ml were offered iron supplementation. Substitution lasted 16 weeks and the donation interval was extended. History collection including iron deficiency-related symptoms, whole blood count and serum ferritin determination was performed at baseline and after 2 and 6 months. Data were recorded prospectively and compared with those of 108 female controls with iron deficiency not receiving iron substitution (retrospective data). RESULTS: Of the 116 participating subjects, 60% completed the programme. Significant results were serum ferritin increase (from a mean value of 7.12 to 25.2 ng/ml), resolution of prostration, fatigue, sleep disturbances, tension in the neck, hair loss and nail breakage. No case of anaemia occurred. Sixty per cent of the women completed the programme and donated blood again. CONCLUSIONS: Targeted iron substitution prevents the development of anaemia and enhances donation return in premenopausal female blood donors with iron deficiency.

Human papillomavirus oncoproteins alter differentiation-dependent cell cycle exit on suspension in semisolid medium.

The suspension of keratinocytes containing episomal forms of the human papillomavirus (HPV)-31 genome in semisolid medium results in the induction of viral late functions. In this study, the suspension in semisolid medium was used to analyze how HPV deregulates the process of cell cycle exit during differentiation. In cells that contain the entire HPV-31 genome, induction of late protein synthesis was found to be linked with the expression of cyclin A. Consistent with analyses in organotypic rafts, the expression of the high-risk E7 oncoprotein alone was sufficient to retain cyclin A expression during suspension-induced differentiation. The cyclin-dependent kinase inhibitors (CKIs) p27 and p57 were found to be up-regulated in normal keratinocytes, as well as in the lines that express the HPV oncoproteins. The up-regulation of these CKIs is coincident with the inhibition of cyclin/cdk activity in normal keratinocytes. Cells expressing E7 were found to retain significant cdk2-associated kinase activity, although it was partially inhibited, coincident with CKI induction. When the phosphorylation state of Rb was examined during differentiation, cells expressing E7 retained phosphorylated forms of Rb, whereas Rb in normal keratinocytes was hypophosphorylated. As previously reported, E7-expressing cells were found to contain less Rb protein than normal keratinocytes. Interestingly, the Rb levels decreased during normal keratinocyte differentiation, and this differentiation-dependent reduction in Rb levels was enhanced by EG and E7 expression. This study identified proteins that may be critical for cell cycle regulation during normal epithelial differentiation and demonstrated that HPV oncoproteins alter their activities.

Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity.

The E6 and E7 proteins from the high-risk human papillomaviruses (HPVs) bind and inactivate the tumor suppressor proteins p53 and Rb, respectively. In HPV-positive cells, expression of E6 proteins from high-risk types results in increased turnover of p53, which leads to an abrogation of p21-mediated G1/S arrest in response to DNA-damaging agents. In contrast, keratinocytes which express E7 alone have increased levels of p53 but, interestingly, also fail to undergo a G1/S arrest. We investigated the mechanism by which E7 bypasses this p21 arrest by using both keratinocytes which stably express E7 as well as U20S cells which stably or transiently express E7. We observed that E7 does not affect the induction of p21 synthesis by p53. While glutathione S-transferase (GST)-E7 bound a low level of in vitro-translated p21, we were unable to detect E7 and p21 in the same complex by GST-E7 binding assays or immunoprecipitations from cell extracts. Furthermore, E7 did not prevent p21-mediated inhibition of cyclin E kinase activity. In keratinocytes expressing E7, increased levels of p53, p21, and cyclin E, as well as increased cyclin E kinase activity, were observed. To determine if this increase in cyclin E activity was necessary for E7's ability to overcome p21-mediated G1/S arrest, we examined U20S cells in which cyclin E levels are not increased in response to E7 expression. U20S cells which stably express E7 were found to initiate DNA synthesis in the presence of DNA-damaging agents despite the inhibition of cyclin E activity by p21. In transient assays, cotransfection of E7 or E2F-1 along with p21 into U20S cells rescued G1 arrest and resulted in S-phase entry, as measured by the ability to incorporate bromodeoxyuridine. These data indicate that E7 is able to overcome G1/S arrest without directly affecting p21 function and likely acts through deregulation of E2F activity.

Activation of papillomavirus late gene transcription and genome amplification upon differentiation in semisolid medium is coincident with expression of involucrin and transglutaminase but not keratin-10.

The life cycle of the papillomaviruses is closely linked to host cell differentiation, as demonstrated by the fact that amplification of viral DNA and transcription of late genes occur only in the suprabasal cells of a differentiated epithelium. Previous studies examining the pathogenesis of papillomavirus infections have relied on the use of organotypic raft cultures or lesions from patients to examine these differentiation-dependent viral activities. In this study, we used a simple system for epithelial differentiation to study human papillomavirus (HPV) late functions. We demonstrate that the suspension of HPV-infected keratinocytes in semisolid medium containing 1.6% methylcellulose for 24 h was sufficient for the activation of the late promoter, transcription of late genes, and amplification of viral DNA. These activities were shown to be linked to and coincide with cellular differentiation. Expression of the late protein E1(wedge)E4 and amplification of viral DNA were detected in the identical set of cells after suspension in methylcellulose. This technique was also used to analyze the differentiation properties of the cells which expressed the late protein E1(wedge)E4. While induction of the spinous layer markers involucrin and transglutaminase was compatible with late promoter induction, expression of the differentiation-specific keratin-10 was shown not to be required for HPV late functions. Interestingly, while the majority of normal human keratinocytes induced filaggrin expression by 24 h, this marker of the granular layer was induced in a smaller subset of HPV type 31 (HPV-31)-positive cells at this time point. The HPV-31-positive cells which expressed filaggrin did not induce the late protein E1(wedge)E4. Use of the methylcellulose system to induce epithelial differentiation coupled with the ability to perform a genetic analysis of HPV functions by using transfection of cloned viral DNA will facilitate the study of the regulation of the papillomavirus life cycle.

Human papillomavirus E7 oncoproteins bind a single form of cyclin E in a complex with cdk2 and p107.

The E6 and E7 proteins of the high-risk human papillomaviruses (HPVs) act coordinately to immortalize human keratinocytes. These viral oncoproteins function by binding and altering the activity of cellular proteins which regulate cell cycle progression. Among the proteins bound by E7 are the retinoblastoma protein, Rb, as well as the related p107 and p130 proteins. In addition, E7 binds cyclin A, which regulates transit through the S and G2/M phases of the cell cycle. In this study, we demonstrate that HPV 18 E7 also associates with cyclin E which controls the G1/S transition. E7/cyclin E complexes were immunoprecipitated from E7-expressing cells as well as from cell extracts using GST-E7 fusion proteins. E7 was found to complex with a single form of cyclin E, and the binding was mediated through p107. Both E7/cyclin E and E7/cyclin A complexes exhibit kinase activity through associated cdk2 proteins which can contribute to phosphorylation of p107. The association of E7 with proteins which regulate transit through the cell cycle may provide an additional mechanism by which infection with human papillomaviruses results in cellular hyperproliferation.

Human papillomavirus type 31 oncoproteins E6 and E7 are required for the maintenance of episomes during the viral life cycle in normal human keratinocytes.

The E6 and E7 oncoproteins of the high-risk human papillomavirus (HPV) types are able to immortalize human keratinocytes in vitro and likely contribute to the development of anogenital malignancies in vivo. The role of these oncoproteins in the productive viral life cycle, however, is not known. To begin to examine these possible roles, mutations in E6 were introduced in the context of the complete HPV 31 genome. Although transfected wild-type HPV 31 genomes, as well as genomes containing an E6 translation termination linker, an E6 frameshift mutation, and a point mutation in the p53 interacting domain were able to replicate in transient assays, only the wild-type genome was stably maintained as an episome. Interestingly, mutant genomes in either the E6 splice-donor site or splice-acceptor site were reduced in replication ability in transient assays; however, cotransfection of E1 and E2 expression vectors restored this function. In a similar fashion, genomes containing mutant HPV 31 E7 genes, including a translation termination mutant, two Rb-binding site mutants, a casein kinase II phosphorylation site mutant, and a transformation deficient mutant, were constructed. Although transient replication was similar to wild type in all of the E7 mutants, only the casein kinase II mutant had the ability to maintain high copies of episomal genomes. These findings suggest a role for E6 and E7 in the viral life cycle beyond their ability to extend the life span of infected cells.