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.

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.

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.

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.

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.