Elfriede Mikeler

Rapid detection and quantification of cell free cytomegalovirus by a high-speed centrifugation-based microculture assay: comparison to longitudinally analyzed viral DNA load and pp67 late transcript during lactation.

BACKGROUND Human cytomegalovirus (HCMV) is reactivated in nearly every seropositive breastfeeding mother during lactation [Lancet 357 (2001) 513]. Conventional tissue culture (TC) and low-speed centrifugation-enhanced microtiter culture methods are not able to detect HCMV from milk during all stages of lactation. OBJECTIVES Development of a sensitive and quantitative microculture technique to describe the dynamics of HCMV reactivation in different milk compartments during lactation. STUDY DESIGN Milk samples were collected longitudinally from seropositive breastfeeding mothers of preterm infants. Native milk samples were separated into fraction 1 (aqueous extract of milk fat), fraction 2 (cell and fat free milk whey) and fraction 3 (milk cells). Each of these fractions was screened qualitatively (TC, nPCR, pp67 late mRNA) and quantitatively (high-speed centrifugation-based microculture, quantitative PCR). RESULTS Prior to low-speed centrifugation-enhanced inoculation, virus particles were concentrated by high-speed centrifugation (60 min at 50,000 x g, 4 degrees C). Using fraction 2 we were able to describe the dynamics of viral reactivation during lactation. We present the course of the quantitative virolactia and DNAlactia and qualitative detection of HCMV pp67 late mRNA in milk whey of four mothers (three transmitters and one non-transmitter). In all these cases virolactia described an unimodal and self limited course. Peak levels of virolactia for transmitters (T1: day 44; T2: day 43; T3: day 50) were closely related the onset of viruria of the corresponding preterm infants (U1: day 39; U2a/U2b: day 44/57; U3: day 60). The courses of viral load coincidence with the courses of DNA load. CONCLUSIONS We present a rapid and highly sensitive microculture method for the quantification of cell free HCMV from milk whey and aqueous extracts from milk fat. Viral reactivation during lactation describes an unimodal course. Our findings have strong implications for quality control of any virus inactivation procedure.

Rapid Simultaneous Detection by Real-Time PCR of Cytomegalovirus UL97 Mutations in Codons 460 and 520 Conferring Ganciclovir Resistance

ABSTRACT Ganciclovir (GCV) resistance is an emerging problem for transplant recipients. A sensitive and rapid real-time PCR approach for simultaneous and semiquantitative detection of human cytomegalovirus (HCMV) UL97 mutations in codons 460/520 was established by LightCycler and confirmed by restriction fragment length polymorphism and sequencing. Results from HCMV laboratory strains were compared with results from 11 GCV-resistant clinical isolates.

Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR : influence of DNA extraction procedures

The diagnostic implications of different procedures of DNA extraction were examined for the detection of HCMV DNA from sera and plasma of immunosuppressed patients. The detection limit of HCMV plasmid DNA from cell free seronegative plasma and serum by limiting dilution nested PCR decreased in the following sequence: phenol/chloroform > NaI-single tube method > proteinase K digestion equal to amplification of native sera and plasma. Nested PCR from native sera and plasma performed well and surpassed the proteinase K method in sensitivity for detection of serum DNAemia. Semi-quantitative determination of HCMV DNA titer present in native sera of immunosuppressed patients did not seem to be correlated to HCMV disease. When compared to the persistence of leukoDNAemia, the viral DNA titer in native plasma could only be observed in the acute phase of HCMV infection, an important phenomenon for diagnostic purposes. Correlation of serum DNAemia to virus culture revealed low positive and high negative predictive values. Predictive values of nested PCR from native sera for HCMV infection were not lower than those following organic DNA extraction. Despite its low correlation to viremia and virus isolation from any site, nested PCR from organic DNA extracts of serum or plasma is the most sensitive diagnostic tool of an ongoing HCMV infection. Additionally, semi-quantitative end point dilution nested PCR from native serum or plasma promises to be a rapid and easy tool for the monitoring of antiviral therapy.

Dynamics of the Emergence of a Human Cytomegalovirus UL97 Mutant Strain Conferring Ganciclovir Resistance in a Pediatric Stem-Cell Transplant Recipient

Stem-cell transplant recipients are at risk of developing ganciclovir-resistant human cytomegalovirus (HCMV) infection caused by mutations in the viral UL97 gene. Knowledge of the relative proportions of coexisting HCMV wild-type and mutant strains may contribute to a better understanding of the dynamics of in vivo mutant strain selection under ganciclovir. Currently, genotype resistance screening for UL97 is routinely performed by restriction fragment length polymorphism detection and sequencing. We present here the longitudinal course of a pediatric recipient of an allogeneic stem-cell transplant infected with a ganciclovir-resistant HCMV strain. EDTA-treated blood samples were analyzed longitudinally. The patient acquired a primary HCMV infection shortly before transplantation and reactivated the virus following allogeneic hematopoietic stem cell transplantation, thus receiving an intensive antiviral treatment schedule. Three different methods for UL97 mutation analysis, restriction fragment length polymorphism detection, sequencing, and a new, real-time PCR approach were performed. In conclusion, for our pediatric patient, during peak viral load, the UL97 wild-type strain predominates, while during clinical deterioration with low viral load, the predominant mutant strain persists.

Investigations on the cause of the nephrotic syndrome in renal amyloidosis

Systematic electron microscopic investigation of glomeruli of 35 patients with renal amyloidosis (grade I–III), among them 26 with the nephrotic syndrome, reveals the following: 1. The extent of the area of basement membrane denuded of its epithelial covering is correlated significantly with the reduction of plasma protein concentration at the time of renal biopsy. 2. In amyloid free regions of the glomerular capillary loops, the foot processes of the epithelial cells remain intact despite the presence of the nephrotic syndrome. From these findings we conclude that the high glomerular protein losses in amyloidosis occur in areas of the basement membrane which are penetrated by amyloid and denuded of their epithelial covering. With increasing number of these lesions per unit area, the permeability of the capillary network for protein increases to a degree which is significantly correlated with the reduced plasma protein concentration at the time of biopsy. The extent of the area of basement membrane denuded of its epithelial covering is correlated significantly with the reduction of plasma protein concentration at the time of renal biopsy. In amyloid free regions of the glomerular capillary loops, the foot processes of the epithelial cells remain intact despite the presence of the nephrotic syndrome. From these findings we conclude that the high glomerular protein losses in amyloidosis occur in areas of the basement membrane which are penetrated by amyloid and denuded of their epithelial covering. With increasing number of these lesions per unit area, the permeability of the capillary network for protein increases to a degree which is significantly correlated with the reduced plasma protein concentration at the time of biopsy.

Dynamics of coexisting HCMV-UL97 and UL54 drug-resistance associated mutations in patients after haematopoietic cell transplantation

BACKGROUND Resistance to antiviral drugs can be a severe problem in transplant recipients. Mutations in the HCMV phosphotransferase-gene (UL97) and the polymerase-gene (UL54) are responsible for resistance against ganciclovir (GCV), cidofovir (CDV) and foscarnet (PFA). Most frequently mutations in the UL97-gene are associated with resistance to GCV. Resistance against PFA and CDV is associated to mutations in the UL54-gene. There are only few reports about multidrug-resistance with mutations in both genes in patients after allogeneic haematopoietic cell transplantation (HCT). OBJECTIVES To asses retrospectively the role of UL97/UL54-mutations for clinical deterioration. STUDY DESIGN We present here three patients after HCT developing multidrug-resistance with coexisting UL97 and UL54-mutations. Genotypical resistance screening was done with restriction-fragment-length-polymorphism (RFLP), sequencing of UL97/UL54, and LightCycler real-time PCR. Phenotyipcal testing was performed by a cell-associated plaque-reduction-assay. Plasma viral-load (VL) was determined longitudinally using Roche Cobas-Amplicor-System (Roche Diagnostics). In one case VL was also correlated to different ratios of coexisting UL97-wildtype and mutant variants. RESULTS All three patients developed multidrug resistant HCMV-infections with one or more UL97 and UL54-mutation detected by RFLP, sequencing and LightCycler-analysis. Two out of three patients showed biphasic VL kinetics with manifestation of UL97 drug-resistance prior/or at peak VL. UL54-mutations emerged also in all three patients either at increasing VL levels of ≥10(5)copies/ml or at peak VL. CONCLUSIONS The development of coexisting HCMV UL97 and UL54-mutations conferring drug-resistance after HCT is not strictly associated with fatal outcome in one of our three patients. Manifestation of drug resistant combined UL97/UL54-mutations occurred prior to a second VL peak under (V)GCV/PFA co-treatment.

Peripolar cells in the avian kidney

Four white leghorn chickens were injected with furosemide (20 mg per kg body weight) three times at 12 h intervals and the kidneys fixed by perfusion after 36 h. Five chickens were injected with DOCA (desoxycortone trimethylacetate, 75 mg per kg body weight) three times at 12 day intervals and the kidneys fixed by perfusion after 36 days. Serial sections from the kidneys of these two groups of birds were made and the number of peripolar cells recorded. These recordings were compared with the number of peripolar cells in four normal, untreated chickens. A significant increase in the number of peripolar cells was recorded in the furosemide treated group. No significant change was seen in the DOCA treated group. However, in the DOCA-group heavily granulated podocytes were found. No distinct morphological difference was found between the granules in the podocytes and the granules in the peripolar cells. A possible lysosomal nature of the peripolar cell granules is discussed.

CD209/DC-SIGN mediates efficient infection of monocyte-derived dendritic cells by clinical adenovirus 2C isolates in the presence of bovine lactoferrin

Adenovirus often causes respiratory infection in immunocompromised patients, but relevant attachment receptors have largely not been defined. We show that the antiviral protein bovine lactoferrin enhances infection of monocyte-derived dendritic cells (MDDC) by adenovirus species C serotype 2 (2C) isolates. Under the same conditions infection of MDDC by human( )cytomegalovirus was reduced. Adenoviral infection was prominently enhanced by bovine but not human lactoferrin, and was not prominently enhanced using blood monocyte-derived macrophages, suggesting that the relevant receptor is expressed on MDDC. Infection of MDDC in the presence of bovine lactoferrin was blocked by mannan, and an antibody to CD209/DC-SIGN but not isotype control or CD46 antibodies. Lastly, U937 macrophages ectopically expressing CD209/DC-SIGN, but not parental U937 cells, were efficiently infected by adenovirus 2C in the presence of bovine lactoferrin. These results may provide a tool, given the high efficiency of infection, to dissect responses by myeloid cells to clinical adenovirus isolates.

Intrafamilial transmission of human cytomegalovirus (HCMV): Long-term dynamics of epitope-specific antibody response in context of avidity maturation

BACKGROUND The role of a special early family formation (PEKiP), which is popular in Germany, as a potential origin of HCMV-transmission to seronegative mothers is not documented. OBJECTIVES To describe the clinical courses, to identify the virological origin and to evaluate a new tool for diagnosis of a cascade of intrafamilial HCMV primary infections. STUDY DESIGN This prospectively analyzed long-term course of HCMV primary infection leading to hospitalization of two family members, included the evaluation of different IgG/IgM/IgG avidity-assays with an epitope-specific recombinant immunoblot-assay. Additionally, neutralization (NT) assays using fibroblast-and epithelial-target cells were performed to correlate NT₅₀ values to avidity maturation. HCMV gN/gO/gB-RFLP-genotyping and phylogenetic analyses were performed using urine viral isolates. RESULTS The clinical courses of the sequentially occurring intrafamilial HCMV primary infections were unusual, leading to hospitalization. Long-term-serology of the mother revealed concordant results for an unimodal IgG-course and a rapid decrease of IgM-indices from week 7 to week 21 p.i. Interestingly, the cut-off definitions for low and high avidity ranged discordantly from 15 to 25 weeks, and from 18 to 42 weeks p.i., respectively. A good correlation was found between the increase of fibroblast-adapted NT50 values and the appearance of high avidity using the epitope-specific immunoblot (>18 weeks p.i.). RFLP-genotyping and sequencing could identify the index patient as member of PEKiP-meetings. CONCLUSIONS PEKiP-meetings with naked babies may be an important source of horizontal HCMV-transmission to seronegative pregnant mothers in Germany. Using epitope-specific immunoblots, persisting HCMVp150-IgM-reactivities and good concordance between high IgG-avidity and increase of fibroblast adapted neutralization capacity were found.

Renin-positive granulated Goormaghtigh cells

SummaryIt is difficult to distinguish between Goormaghtigh cells (G-cells) and media cells of the glomerular arterioles at the border of the Goormaghtigh cell field. Consequently, it has been unclear whether renin-positive G-cells are normally present and also whether renin-producing cells are recruited from the pool of renin-negative G-cells upon stimulation of the renin-angiotensin system (RAS). In the present study, immunohistochemical and electron-microscopic experiments have been carried out on serially sectioned kidney biopsies from four patients with pseudo-Bartter syndrome. The results strongly suggest that with longlasting stimulation of the RAS all renin-negative (“secretory resting”) G-cells are ultimately converted into renin-producing granular cells.