Label-free protein quantification of sodium butyrate treated CHO cells by ESI-UHR-TOF-MS.

Effects of butyrate on CHO producer cells are contradictory, promoting productivity and at the same time repressing proliferation. Though in previous omics studies the background of butyrate impact on producer cells has been investigated, the knowledge about the mechanism is still very limited. As previous proteomic results on this field are mainly based on 2DE-gels, we conducted a label-free MS quantification, based on fast high resolution ESI-MS and a straight forward software solution, to gain insight in shifted cellular processes of CHO cells 25h after butyrate treatment. 118 proteins or subunits with significantly altered abundances were identified suggesting changes in carbohydrate, protein metabolic and cell cycle processes. Effects of butyrate on the nucleosome assembly as a known direct epigenetic influence on HDAC activity turned out to be unexpectedly fast and persistent, as confirmed by Western blots of histone-H4 acetylation. Contradictory to increased cell specific productivity, most elements of protein metabolism exhibited decreased levels after butyrate treatment. In comparison to published results some overlap of our label free MS data could be observed but also apparently diverging findings, showing the need for complementary omics techniques for a holistic view on cellular processes such as response to butyrate.

A Rare Clinical Course of Seronegative Pulmonary-Renal Syndrome.

Purpose. Pulmonary-renal syndrome (PRS) is characterized by diffuse alveolar hemorrhage and rapidly progressive glomerulonephritis mainly due to autoimmune etiologies. Seronegative PRS is a challenging entity to the clinician, since early diagnosis may be missed leading to delayed appropriate treatment. Materials and Methods. We present the clinical course of a 77-year-old patient who was admitted under the suspected diagnosis of pneumogenic sepsis and septic renal failure with fever, dyspnea, and elevated CRP levels. The diagnosis of pulmonary-renal syndrome was initially missed because of the absence of autoantibodies in all serological findings. Results. Despite delayed initiation of immunosuppressive therapy and a prolonged period of dialysis and extracorporeal membrane oxygenation the patient recovered well and was released to a rehabilitation center with nearly normalized creatinine levels. The diagnosis of PRS was established by renal biopsy. Conclusion. This case illustrates the important differential diagnosis of seronegative pulmonary-renal syndrome in patients with pulmonary and renal impairment.

Vasculitic purpura with antineutrophil cytoplasmic antibody-positive acute renal failure in a patient with Streptococcus bovis case and Neisseria subflava bacteremia and subacute endocarditis.

Subacute bacterial endocarditis is frequently associated with extracardiac manifestations and renal failure. Clinical variety of endocarditis manifestation is wide and has the potential to mimic vasculitis. Whereas Streptococcus bovis is often isolated and associated with colonic tumors, Neisseriaceae are rarely found. An association of subacute bacterial endocarditis and antineutrophil cytoplasmic antibodies has been described. We report on a 62-year-old man who was admitted to our hospital with acute oliguric renal failure and a nonpruritic purpural rush without fever. Antineutrophil cytoplasmic antibody diagnostic revealed perinuclear staining with a titre of 1 : 512 and antiproteinase-3 specificity. Immune complex-mediated glomerulonephritis without extracapillary proliferation was diagnosed in renal biopsy. Finally, blood cultures became positive for Streptococcus bovis and Neisseria flava. Echocardiography showed mobile vegetations on tricuspid valve. Under treatment with penicillin G and gentamicin, skin efflorescences and renal function recovered, but vegetations increased. A colonic tumor could be excluded, a disastrous dental status may have been a predisposal factor. When classical findings of subacute bacterial endocarditis are less clear, the presence of renal failure and antineutrophil cytoplasmic antibodies in absence of fever may lead to misdiagnosis and deleterious immunosuppressive therapy. Neisseria subflava, an upper respiratory tract commensal, may cause subacute bacterial endocarditis without typical symptoms.

Polarized expression of Tamm-Horsfall protein by renal tubular epithelial cells activates human granulocytes.

In renal bacterial infections granulocytes are of major importance in the primary immune defense against invading pathogens. However, the mechanisms of granulocytic activation in renal interstitial invasion have not been clarified. Renal tubular epithelial cell mechanisms inducing granulocytic activation and bacterial killing may include tubular cell expression of Tamm-Horsfall protein (THP), a urinary protein that is known to enhance cytokine expression in monocytes. We studied the role of THP in granulocytic activation. A strong binding of THP to human granulocytes was demonstrated by fluorescence-activated cell sorter analysis. Urinary THP and supernatants of THP-expressing cultured tubular epithelial cells (MDCK) enhanced interleukin-8 (IL-8) expression by human granulocytes. Renal tubular cells growing polarized on polycarbonate membranes were used to study apical versus basal THP expression. By electron microscopy THP immunoreactivity was exclusively found on the apical surfaces of tubular cells and was absent on the basolateral cell membrane. In the apical cell culture compartment we found significantly more stimulatory activity for granulocytic IL-8 expression. CD62L, a selectin less expressed in activated granulocytes, was decreased in granulocytes incubated with urinary THP and in supernatants of THP-producing renal tubular cells but not in supernatants from THP-negative cells. Again, the effect on CD62L expression was found only in apical culture media and was absent in the basal compartment. In summary our data give evidence that renal tubular cell THP expression may be relevant in kidney diseases since THP is a potent activator of human granulocytes. The regulation of apical versus basal THP expression and release in vivo may be crucial in the induction of the inflammatory response, e.g., in bacterial renal diseases.

Hemolytic anemia after kidney transplantation: case report and differential diagnosis.

A 58-year-old woman presented with hemolysis and thrombocytopenia 2 weeks after receiving a kidney graft. Hemolytic uremic syndrome was initially suspected, because in addition to hematological changes the graft function was missing. Unexpectedly, the results of the direct antiglobulin test became positive (4+), which is not normally observed in the hemolytic uremic syndrome. Differentiation of the eluted antibodies revealed anti-rhesus D specificity, which had to be interpreted either as an autoantibody of patient's origin or, hypothetically, as a "graft versus host" antibody of donor origin. Gm- and Km allotyping of these antibodies demonstrated a pattern which differed from the patient's but was identical to that of the kidney donor. Therefore hemolysis could be explained unambiguously by "graft versus host" antibodies. Whether the thrombocytopenia was also due to an immune process was not clear, although some evidence favors this hypothesis. Immunosuppressive treatment remained unchanged and several red blood cell transfusions were necessary before reactivity of the direct antiglobulin test diminished and became negative 7 weeks after kidney transplantation. The occurrence of hemolysis in the early posttransplantation period should thus draw attention to the possibility of "graft versus host" antibodies directed against red cells. Concomitant thrombocytopenia may occur. Donor screening for irregular erythrocyte antibodies should be performed whenever solid organ transplantation is intended.

Patients at risk for development of posttransplant lymphoproliferative disorder: plasma versus peripheral blood mononuclear cells as material for quantification of Epstein-Barr viral load by using real-time quantitative polymerase chain reaction.

BACKGROUND: Early diagnosis of Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder (PTLD) is required to detect a stage of disease that is more likely to respond to treatment. Elevated levels of EBV DNA were found in peripheral blood of patients at the onset of PTLD. METHODS: To compare plasma and peripheral blood mononuclear cells (PBMCs) as material for real-time quantitative polymerase chain reaction (RQ-PCR) measurement of Epstein-Barr viral load, we used two sets of primers and probes specific for the BAM HI-K or BAM HI-W region of the EBV genome. RESULTS: Patients with PTLD had a median viral load of 19,200 EBV genomes/microg DNA (n=9) or 3,225 EBV genomes/100 microl plasma (n=5), being significantly higher compared with immunosuppressed patients with primary (n=9) or reactivated (n=20) EBV infection or immunosuppressed patients without serological signs of active EBV infection (n=67) (P<0.001). Hence, a value of greater than 5,000 EBV genomes/microg PBMC DNA was considered as a diagnostic parameter for PTLD with a sensitivity and specificity of 1.00 or 0.89, respectively. When plasma was analyzed, however, a value of greater than 1,000 EBV genomes/100 microl plasma had both a sensitivity and specificity of 1.00 for the diagnosis of PTLD. During remission of PTLD, viral load was more effectively cleared in plasma compared with PBMCs. In plasma of nonimmunosuppressed individuals, even a qualitative detection of EBV-related sequences was sensitive and specific for the diagnosis of primary EBV infection, whereas for analysis of PBMC DNA a quantitative parameter had to be considered to differentiate healthy individuals (< 100 EBV genomes/microg PBMC DNA) from patients with primary EBV infection (>100 EBV genomes/microg PBMC DNA). CONCLUSION: Although both PBMCs and plasma were useful as material for EBV-specific RQ-PCR in immunosuppressed patients and nonimmunosuppressed individuals, the specificity of analysis seemed to be higher if plasma was taken for analysis.

Normalized quantification by real-time PCR of Epstein-Barr virus load in patients at risk for posttransplant lymphoproliferative disorders.

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.

Real-time polymerase chain reaction (RQ-PCR) for the monitoring of Epstein-Barr virus (EBV) load in peripheral blood mononuclear cells.

Measurement of Epstein-Barr virus (EBV) load is useful in peripheral blood for detecting primary and reactivated EBV-infections especially in immunosuppressed patients being at high risk for developing posttransplant lymphoproliferative disorder. For quantification of EBV DNA in peripheral blood of patients two real time polymerase chain reaction (RQ-PCR) assays were developed detecting sequences specific for the BAM HI-W and BAM HI-K region of EBV. In order to determine the optimal material of peripheral blood for RQ PCR analysis, DNA preparations of whole blood, peripheral blood mononuclear cells (PBMC) and B cells from 11 healthy, EBV-seropositive individuals were analysed in parallel and compared with regard to efficiency and sensitivity. While in whole blood preparations inhibitors of RQ PCR were detected influencing sensitivity, analysis of B cells being most sensitive is limited by being too labour intensive. In contrast, analysis of DNA preparations of PBMCs is sensitive enough to frequently detect EBV-specific sequences in all individuals tested and the preparation of PBMCs itself needs only a reasonable time. Thus, longitudinal monitoring of EBV load in peripheral blood of patients is possible by RQ-PCR, the optimal material for analysis being PBMCs.

The primary and memory immune response to Epstein-Barr virus infection in vitro is characterized by a divergent production of IL-1beta/IL-6 and IL-10.

Reactivation of latent Epstein-Barr virus (EBV) infection is considered to exert substantial immunomodulating activities. Little is known about EBV's modulating activities on cytokine production upon primary, in contrast to reactivated, infection. Therefore, we investigated the cytokine production of latently infected EBV-positive (EBV-pos) adults upon in vitro EBV stimulation compared to nonimmune age-matched EBV-negative (EBV-neg) donors. Production of interleukin (IL)-1beta and IL-6 was strongly decreased in peripheral blood mononuclear cell (PBMC) cultures of EBV-pos adults; in contrast, IL-10 and IL-1 receptor antagonist exhibited significantly higher levels. As expected, interferon (IFN)-gamma production was almost exclusively observed in EBV-pos donors; it was accompanied by a significantly higher IL-12 synthesis. Experiments employing T cell-depleted PBMC showed similar cytokine levels between EBV-pos and EBV-neg individuals suggesting that reactivation of EBV-specific memory T cells was responsible for the divergent cytokine profiles. Production of viral IL-10 was excluded as a reason for higher IL-10 levels in EBV-pos individuals. In conclusion, our results do not appear to relate to any primary immunological differences between EBV-neg and EBV-pos adults, but show that the EBV-specific memory response to latent EBV infection is characterized by some anti-inflammatory effects. This might be of relevance upon reactivation of latent EBV infection in vivo and provides further evidence that EBV infection acts in an immunomodulating fashion.

Association between clinical disease activity and Epstein-Barr virus reactivation in MS.

OBJECTIVE: To assess the potential significance of Epstein-Barr virus (EBV) reactivation in disease activity in MS patients. METHODS: The prevalence of antibodies against herpes simplex virus type 1 (HSV-1), HSV-2, EBV, and cytomegalovirus was determined in a group of 108 MS patients and in 163 healthy control subjects. Sera were analyzed using combinations of novel assay systems employing highly purified viral and recombinant antigens. In addition, PCR for the detection of EBV DNA was performed in serial samples. RESULTS: In contrast to the control populations, antibodies against EBV were present in 100% of MS patients. Among the tested human herpesviruses, this high extent of seropositivity was only found for EBV. Primary infection was found exclusively in the control group (3.7%), whereas serologic evidence of EBV reactivation was seen in MS patients (13. 9%) as well as control subjects (17.2%). There was no temporal coincidence between EBV reactivation and disease activity in MS patients. However, in 19 patients followed monthly for 1 year, active viral replication as measured by increased immunoglobulin (Ig) M and IgA responses to EBV early antigens (p54 + p138) and positive serum DNA was seen in 72.7% of patients with exacerbations during the study period and in none of the patients with clinically stable disease. CONCLUSIONS: The results demonstrate an association between EBV reactivation and disease activity in MS patients over time, and suggest that EBV might play an indirect role in MS as an activator of the underlying disease process.

The influence of naturally occurring heterophilic anti-immunoglobulin antibodies on direct measurement of serum proteins using sandwich ELISAs.

Sandwich ELISAs have become a widely used method for the quantitative detection of serum proteins. However, they can be biased by a variety of interfering substances. As reported recently, we observed false-positive levels of interferon (IFN)-alpha and -beta in up to 27% of sera from healthy blood donors using commercial ELISAs. We now demonstrate that two different groups of naturally occurring heterophilic antibodies (IgG-type) are responsible for these titers. Group I (representing 85% of positive samples) binds to the Fab region of IgG from goat, mouse, rat, horse, and bovidae (but not rabbit). Group II (15%) recognizes an epitope in the Fc region of mouse, horse, bovine, and rabbit (but not goat or rat) immunoglobulins. The antibodies did not crossreact with human IgG subclasses but contributed to false-positive IgG rheumatoid factor levels obtained using a commercially available ELISA. To investigate the susceptibility of assays to these artifacts, various combinations of capture and detection antibodies have been tested. On this basis, we defined the relative risks that standard ELISAs might be influenced by heterophilic anti-immunoglobulin antibodies. In general, assays that use monoclonal antibodies for both capture and detection are less susceptible than others which include at least one polyclonal antiserum. However, only systems utilizing rabbit F(ab')(2) fragments have been found to be immune to this interference.

Altered prevalence and reactivity of anti-Epstein-Barr virus antibodies in patients with multiple sclerosis.

In this study, the prevalence and reactivity of anti-Epstein-Barr virus (EBV) antibodies were investigated in 107 patients with multiple sclerosis (MS) in comparison to age- and gender-matched healthy controls from a north German state. We found a significant 100% EBV-seropositivity and a significant lack of primary EBV infections in the MS group, indicating that all MS patients are infected with EBV before the development of MS. Although there were no differences in reactivities of EBV-specific anti-early antigen (EA)-immunoglobulin G (IgG), -IgM, and -IgA antibodies between each group, MS patients had significant lower anti-Epstein-Barr nuclear antigen (EBNA)1-IgG antibody titers as a possible serological sign for a defective control of the persistent latent EBV carrier state and EBV reactivations. Longitudinal studies of MS patients are necessary to further determine the implications of EBV reactivations on the course and disease activity of MS.

Failure to detect antiviral activity in serum and plasma of healthy individuals displaying high activity in ELISA for IFN-alpha and IFN-beta.

The presence of constitutively produced interferon (IFN)-alpha in the blood of healthy individuals has been the subject of contradictory discussions for years. Immunologic as well as biologic test procedures have demonstrated striking differences regarding serum IFN-alpha under physiologic conditions. We investigated the presence of immunoreactive IFN-alpha in serum samples of 923 healthy blood donors by means of a widely used commercially available ELISA. Of these, 254 (27.5%) exhibited detectable serum IFN-alpha levels. The sera of 85.1% of these people also contained IFN-beta. Both IFN were also demonstrated in EDTA-anticoagulated plasma. However, none of these samples exhibited any antiviral effect on human A549 lung carcinoma cells challenged with encephalomyocarditis virus. Samples with high IFN-alpha ELISA activity did not abolish the antiviral action of added natural IFN-alpha, thus excluding IFN-alpha inhibitory factors. The experiments suggest that the detected compounds probably did not represent IFN-alpha but were the result of a cross-reaction with unknown serum components. A variety of disorders has been associated with elevated serum IFN-alpha levels that in most cases were detected by ELISA. In view of our data, these findings need to be carefully reevaluated. For the purpose of monitoring IFN-alpha levels in therapy of atopic, autoimmune, or malignant disorders, an appropriate detection system for IFN-alpha is advisable.

Analysis of Epstein-Barr virus (EBV) receptor CD21 on peripheral B lymphocytes of long-term EBV- adults.

Primary infections with EBV are rarely observed after the age of 20. Some individuals even remain seronegative all their lives. Previously, a lack of EBV receptors on B cells of persistently EBV- adults was described as a reason for long-term EBV-seronegativity. The present study examined the CD21 receptor status of 20 repeatedly EBV- healthy adults and 32 EBV+ volunteers by means of flow cytometry. CD21 molecules on the surface of CD19+ B cells were quantified using anti-IgG-coated microbeads. The percentage of CD19+/CD21+ B lymphocytes was slightly lower in the peripheral blood of EBV- donors, but the CD21 antibody binding capacity on CD19+ B cells showed no significant differences between EBV- and EBV+ adults. In vitro studies showed an equally good EBV transformability of peripheral B lymphocytes of EBV- and EBV+ donors. Since HLA-DR was recently described as a co-receptor for EBV infection of B cells, we also determined HLA-DRB1 alleles in the EBV- group. We found a significant negative association of EBV-seronegativity with HLA-DR13 in comparison with 111 healthy blood donors. In summary, a biologically significant lack of the EBV receptor CD21 on peripheral B lymphocytes of persistently EBV- adults was excluded as a reason for long-term EBV-seronegativity.

Assignment and Power Series Analysis of the FIR Fourier Transform Spectrum of Cyanamide Using a Multimolecule Ritz Program.

The "Ritz" program, originally written for the analysis of the Fourier transform spectra of the methanol isotopomers and presented in previous papers, has been extended in order to analyze the spectra of other molecules. This program evaluates the term values involved in the assigned transitions by the Rydberg-Ritz combination principle, and can tackle such perturbations as Fermi-type resonances or Coriolis interactions. As a first application of the extended version, we present an investigation of the Fourier transform spectrum of cyanamide between 25 and 980 cm-1. More than 16 000 lines have been assigned. Our Ritz database now comprises a list of more than 19 000 assigned lines (including of the microwave and FIR lines available in the JPL database) and more than 3900 term values. All of the lines presented in this paper correspond to transitions within the ground and first excited inversion levels of the ground vibrational state of the small-amplitude modes. Copyright 1998 Academic Press.

Characterization of the novel, pediatric Hodgkin disease-derived cell line HKB-1.

BACKGROUND: A novel Hodgkin disease (HD)-derived cell line, designated HKB-1, was established from pulmonary HD of nodular sclerosing subtype of a 14-year-old-girl. PROCEDURE AND RESULTS: Immunophenotypically, HKB-1 cells are of B cell in phenotype, being also positive for markers CD15, CD25 and CD30. Transformation of the cell line by Epstein-Barr virus (EBV) was excluded by failure to detect EBV antigens and EBV DNA in cultured cells. Chromosome studies of HKB-1 showed a pseudodiploid karyotype with complex clonal structural aberrations. The detection of a monoclonal immunoglobulin heavy chain (IgH) gene rearrangement confirmed the derivation of the HKB-1 from the B cell lineage and its monoclonality. HKB-1 produced high amounts of interleukin (IL)-6 as detected in culture supernatant whereas no secretion of IL-1 beta, IL-2, IL-4, IL-10, IL-12 or interferon (IFN)-gamma was detected. CONCLUSIONS: Our studies indicate that this cell line is of tumor origin.

Immunologic properties of Epstein-Barr virus-seronegative adults.

Epstein-Barr virus (EBV) seronegativity is rare in people > 20 years old. However, some persons remain EBV-seronegative for nearly their whole lives. The aim of this study was to examine properties of the immune system of EBV-seronegative adults that could contribute to long-term EBV seronegativity. Therefore, differential blood cell counts and lymphocyte subpopulations were determined, and the production of interferon (INF)-alpha and -gamma and interleukin (IL)-6 and -2 in a whole blood assay was investigated. Whereas no differences in the distribution of lymphocyte subpopulations between EBV-seronegative and -positive adults were found, a significant higher percentage of monocytes in EBV-seronegative adults was observed. Significantly more IFN-alpha and IL-6 were detected in culture supernatants of EBV-seronegative persons after stimulation with Newcastle disease virus. In contrast, no differences in the induction of the lymphokines IFN-gamma and IL-2 were seen. These data suggest that faster and higher production of IFN-alpha and IL-6 amy protect EBV-seronegative adults against EBV infection.