Characterization of CD8+ T-cell response in acute and resolved hepatitis A virus infection.

BACKGROUND & AIMS: In contrast to the infection with other hepatotropic viruses, hepatitis A virus (HAV) always causes acute self-limited hepatitis, although the role for virus-specific CD8 T cells in viral containment is unclear. Herein, we analyzed the T cell response in patients with acute hepatitis by utilizing a set of overlapping peptides and predicted HLA-A2 binders from the polyprotein. METHODS: A set of 11 predicted peptides from the HAV polyprotein, identified as potential binders, were synthesized. Peripheral blood mononuclear cells (PBMCs) from patients were tested for IFNγ secretion after stimulation with these peptides and ex vivo with HLA-A2 tetramers. Phenotyping was carried out by staining with the activation marker CD38 and the memory marker CD127. RESULTS: Eight out of 11 predicted HLA-A2 binders showed a high binding affinity and five of them were recognized by CD8+ T cells from patients with hepatitis A. There were significant differences in the magnitude of the responses to these five peptides. One was reproducibly immunodominant and the only one detectable ex vivo by tetramer staining of CD8+ T cells. These cells have an activated phenotype (CD38hi CD127lo) during acute infection. Three additional epitopes were identified in HLA-A2 negative patients, most likely representing epitopes restricted by other HLA-class I-alleles (HLA-A11, B35, B40). CONCLUSIONS: Patients with acute hepatitis A have a strong multi-specific T cell response detected by ICS. With the tetramer carrying the dominant HLA-A2 epitope, HAV-specific and activated CD8+ T cells could be detected ex vivo. This first description of the HAV specific CTL-epitopes will allow future studies on strength, breadth, and kinetics of the T-cell response in hepatitis A.

Human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis show the imprints of an antigen-dependent process of somatic hypermutation and clonal selection.

The persistent presence of rheumatoid factors (RFs) in the circulation is a characteristic phenomenon in patients with rheumatoid arthritis (RA). Recent data indicate that RFs associated with seropositive RA are derived from terminally differentiated CD20-, CD38+ plasma cells (PCs) present in synovial fluids of the inflamed joints. These cells were shown to secrete RFs actively and are thought to originate from germinal centre (GC)-like structures present in the inflamed synovium. To obtain a representative image of the structural properties of IgM and IgG RFs associated with RA, phage antibody display libraries were constructed from CD38+ PCs isolated from the inflamed joints of RF-seropositive patients with RA. Subsequently, human IgG Fc-binding monoclonal phage antibodies were selected and analysed. The data suggest that RA-associated RFs are encoded by a diverse set of VL and a more restricted set of VH regions. VH gene family usage of PC-derived IgM- and IgG-RFs was found to be restricted to the VH1 and 3 gene families, with a preference for VH3, and many different VL genes were shown to contribute to RF specificity. Clonally related VH as well as VL sequences were identified, based on the presence of identical CDR3 regions and shared somatic mutations. In this B cell selection process base-pair substitutions as well as deletions of triplets in CDR regions, leaving the transcripts in frame, were involved. Together, these data provide further evidence for an Ag-driven immune response in the terminal differentiation into RF-producing PCs in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a GC reaction.

Polyreactivity of human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis.

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20-, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells.

Presence of a population of CD20+, CD38- B lymphocytes with defective proliferative responsiveness in the synovial compartment of patients with rheumatoid arthritis.

OBJECTIVE: To provide a comprehensive understanding of the humoral immune response that takes place at the site of inflammation in rheumatoid arthritis (RA), we studied the functional properties of synovial B cells. In particular, the response to various modes of mitogen stimulation was investigated. METHODS: Purified synovial fluid (SF) B cells were cultured in the presence of CD40 ligand (CD40L)-expressing fibroblasts and cytokines, activated T cells, or phorbol myristate acetate (PMA)/ionomycin. Proliferation was determined by 3H-thymidine incorporation. Release of intracellular calcium was studied by flow cytometry. RESULTS: The inflamed joints of RA patients contained a population of CD20+,CD38- B cells with dramatically impaired mitogen responsiveness. Although the Ig-producing capacity was intact, these cells failed to proliferate in response to (a) CD40 in the presence of interleukin-2 (IL-2) and IL-10, (b) activated T cells, or (c) stimulation via the B cell receptor. Moreover, SF CD20+,CD38- B cells revealed a defective B cell receptor-induced Ca2+ influx, reminiscent of anergic B cells. Release of intracellular Ca2+ by ionomycin in the presence of the protein kinase C activator PMA did not restore the proliferative capacity. These findings indicate blockades in the proximal and distal intermediates involved in mitogen signaling. CONCLUSION: SF CD20+,CD38- B cells have functionally impaired proliferative responsiveness. The capacity of these cells to respond to activation by the production of Ig supports the notion that these cells might serve as Ig-producing effector cells and, as such, play a role in the pathophysiology of RA.

Differential requirements for induction of total immunoglobulin and physiological rheumatoid factor production by human peripheral blood B cells.

Rheumatoid factors (RFs) are autoantibodies directed against the Fc part of IgG. Considerable evidence exists that there are two classes of RFs, pathological and physiological. Whereas pathological RFs are associated with disease, physiological RFs are considered to be a normal component of the immune response. RF(+) precursor B cells present as part of the B cell repertoire of healthy individuals are held responsible for the production of physiological RFs, which is a transient phenomenon with a clear correlation with an initiating stimulus such as immunization or exposure to an infection. Here we demonstrate a difference in the regulatory control of total Ig and RF production by peripheral blood (PB) B cells of both healthy controls (HC) and patients with rheumatoid arthritis (RA). Highly purified B cells from HC and patients with RA were cocultured with T cells stimulated with immobilized anti-CD3 mAb. Similar to IgM production, IgM-RF production was shown to be dependent on CD40 cross-linking. However, activation of PB B cells in the CD40 system in the presence of IL-2, IL-4, IL-10, combinations of these cytokines or supernatant of anti-CD3-stimulated T cells failed to induce detectable IgM-RF, whereas total IgM production was considerable. From these results we conclude that conditions to activate physiological RF(+) B cells require additional contact besides CD40--CD40L interactions between T and B cells. Since the requirements for RF production were similar using PB B cells from HC and patients with RA it is suggested that the regulatory properties of RF(+) precursors in the PB B cell compartment is equal among these groups. Together, these results indicate that conditions for the induction of total Ig and physiological RFs are different.

Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis.

OBJECTIVE: To understand the regulation of anti-citrulline-containing peptide antibody (anti-CCP) production in rheumatoid arthritis (RA), production of anti-CCP by B cells derived from peripheral blood (PB), bone marrow (BM), and synovial fluid (SF) was examined. METHODS: Purified PB and SF B cells were isolated by negative selection and then cultured in the absence or presence of L-CD40 ligand cells and interleukin-10 or anti-CD3-activated T cells. Total IgM and IgM-anti-CCP were detected after 14 days of culture by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to analyze the frequency of cells that spontaneously produced IgM-anti-CCP in BM and SF B cells. RESULTS: IgM-anti-CCP autoantibodies were induced in PB B cells from healthy controls and RA patients following coculture with activated T cells or application of the CD40 activation system, whereas no production could be detected when PB B cells were cultured in the absence of a stimulus. SF and BM B cells from anti-CCP-seropositive RA patients, but not anti-CCP-seronegative patients, actively produced IgM-anti-CCP without stimulation. The frequency of spontaneous production of IgM-anti-CCP among the IgM-secreting cells ranged from 2.2% to 25%. CONCLUSION: These results indicate the presence of B cell precursors for anti-CCP autoantibodies that are able to produce antibodies upon stimulation in the PB B cell repertoire of healthy controls and patients with RA. In contrast, B cells that actively secreted anti-CCP were specifically present in the BM and SF compartment of anti-CCP-seropositive RA patients. The local presence of anti-CCP-secreting cells in the inflamed joints provides evidence for an antigen-driven maturation of CCP-specific B cells at the site of inflammation in RA.

Management of acute uncomplicated urinary tract infections in general practice in the south of The Netherlands.

There is debate about the ideal diagnostic procedure for urinary tract infections (UTIs) in general practice. The aim of this study was to evaluate nitrite and leucocyte esterase strip test procedures in general practice patients, and to relate the results to the decision of the general practitioner to prescribe antibiotic therapy. A total of 292 female patients from eight general practices in the Maastricht area, who were aged 12 years or over with complaints suggesting UTI, were included in the study. All eight practices tested fresh urine samples using the nitrite strip test, and seven also used the leucocyte esterase strip test. The positive predictive value of the nitrite test was greater than the leucocyte test. Antibiotic therapy was nearly always prescribed when either or both of these tests were positive. Bacterial culture was positive in 159 (59%) cases, although treatment was started in 70 (27%) cases where there was an absence of significant bacteruria. It was found that the choice of agent used to treat the patient was related to the antibiotic susceptibility of the uropathogens that were isolated.

Regulation of synovial B cell survival in rheumatoid arthritis by vascular cell adhesion molecule 1 (CD106) expressed on fibroblast-like synoviocytes.

OBJECTIVE: B lymphocytes accumulate in the inflamed joints of patients with rheumatoid arthritis (RA) and are responsible for production of high amounts of (auto)-antibodies. The aim of this study was to determine the capacity of fibroblast-like synoviocytes (FLS) to contribute to the accumulation of synovial fluid (SF) B cells by extending their life span. METHODS: Highly purified SF B cells were cultured with FLS in the presence or absence of blocking antibodies directed against cell adhesion molecules, and cell viability was determined after various time intervals by trypan blue, annexin V, propidium iodide, or Hoechst staining. Phenotypic characterization of peripheral blood and SF B cells and FLS was carried out by flow cytometry. RESULTS: Synovial B cells, which consist predominantly of memory B cells and plasma cells (PC), undergo spontaneous cell death by apoptosis upon removal from their in vivo environment, despite expression of Bcl-2. Coculture with FLS rescued synovial B cells from apoptosis in a cell contact-dependent manner. Blocking studies using monoclonal antibodies demonstrated a role for the molecular interaction of SF B cells with vascular cell adhesion molecule 1 (VCAM-1; CD106) in FLS-induced survival. The ability of FLS to induce SF B cell survival was not related to the rheumatoid origin since FLS from non-RA patients had similar properties. CONCLUSION: These findings indicate a crucial role for FLS in the survival of synovial B cells at the site of inflammation in RA through the interaction with VCAM-1 expressed on FLS. Consequently, memory B cells and PC accumulation arise and persist not only as a result of maturation and recruitment of these cells, but also by active prevention from cell death by the microenvironment.

Functional analysis of rheumatoid factor-producing B cells from the synovial fluid of rheumatoid arthritis patients.

OBJECTIVE: To understand the regulation of rheumatoid factor (RF) production in rheumatoid arthritis (RA), we studied IgM-RF production by B cells isolated from the synovial fluid (SF). METHODS: Highly purified SF and peripheral blood (PB) B cells were isolated by negative selection in a fluorescence-activated cell sorter (FACS) and then cultured with either L cells, CD40 ligand (CD40L)-transfected L cells, or type B synoviocytes in the presence or absence of interleukin-2 (IL-2), IL-4, or IL-10. Total IgM and IgM-RF were detected after 14 days by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to detect cells that spontaneously produced immunoglobulin. SF B cells were also phenotypically characterized by FACS analysis. RESULTS: Terminally differentiated CD20-,CD38+ synovial plasma cells (PC) present in the SF of RA patients secreted IgM-RF in the absence of a stimulus. IgM-RF production markedly increased when SF B cells were cultured in the presence of type B RA synoviocytes together with IL-10, but independently of CD40-CD40L interaction. Although CD20-,CD38+ PC could also be demonstrated in SF B cells from patients with other forms of arthritis, IgM-RF production was restricted to the SF B cell cultures of patients with seropositive RA. The frequency of IgM-RF-producing cells among IgM-producing PC in patients with seropositive RA was estimated to be as much as 50%. CONCLUSION: These data demonstrate that terminally differentiated CD20-,CD38+ IgM-RF-producing B cells are specifically present in the inflamed joints of patients with seropositive RA. There is evidence that the local environment in the rheumatoid joint favors RF production. The relatively high frequency of IgM-RF PC in the SF B cell population provides evidence of a dominant RA-specific antigen-driven response in the development of the synovial PC repertoire.

[Graduate education for specialist in surgery in the USA--aspects for the young German and American physician]. / Die Weiterbildung zum Facharzt für Chirurgie in den USA--Aspekte für den jungen deutschen und amerikanischen Arzt.

Due to high standards in clinical practice and outstanding research opportunities in the USA, German residents wish to do a part of their surgical education in an American teaching hospital. Currently, German applicants must have an unrestricted license and a valid Standard ECFMG Certificate. For this certificate they must have passed Step I and II of the United States Medical Licensing Examination, which also includes an English test. The examination requirements are about to be changed. A 1-year participation in one of the 267 surgical residency programs is generally not possible. In Germany, American residents have the opportunity to apply for a supervised training year without passing the German medical exams. At present, there are no clinical exchange programs in surgery between Germany and the USA. Therefore, the German and American College of Surgeons should develop an exchange program for residents in surgery.

[Repeated internal urethrotomy in male urethral stricture]. / Die Urethrotomia interna der männlichen Harnröhrenstriktur bei mehrfacher Anwendung.

The results of further treatment of 40 patients with recurrence of urethral stricture following internal urethrotomy are assessed. Repeat internal urethrotomy was often successful even in cases presenting with extensive stricture, multiple stenoses or stenosis in an unfavourable site. Altogether 47% of the patients remained recurrence-free after the second attempt at internal urethrotomy. Open surgery was necessary in 3 patients. In old or unfit patients who could not be subjected to open surgery internal urethrotomy was performed up to 5 or even 6 times. This operation is often preferred by patients to conservative forms of treatment such as bougienage or an indwelling catheter. It is tolerated well, even on a repeated basis and the complication rate is low.

[Success of internal urethrotomy as the 1st operation in male urethral stricture]. / Der Erfolg der Urethrotomia interna als Erstoperation bei Harnröhrenstriktur des Mannes.

A follow-up examination was possible on 179 out of 226 patients operated on for urethrostenosis using sight urethrotomy according to Sachse. The results are classification according to the observation period, ranging from 3 months to 3 years. The most important criteria of a successful operation were lack of discomfort and a rise in uroflow to over 10 ml. Relapses occur more frequently than average in patients who had recidive infections of the urinary tract before the operation. Where the operation was successful the urinary tract infection almost always subsided. Recidive strictures occurred frequently in patients over 70 years. However, internal urethrotomy is often the only suitable method of operation at that age. Long prior bouginage makes prognosis worse. In terms of aetiology, iatrogenic stenoses give the worst results, bulbar ring stenoses, the best. In cases of long stenoses, particularly in the penile part of the urethra, the results of the operation are clearly worse than average, so that another method of operation should be considered, especially if the patients are young.

[Treatment of Wilms' tumor (author's transl)]. / Therapie des Wilms-Tumors.

Uniform treatment based on the therapeutic approach of the 1st and 2nd US National Wilms' Tumor Study was decided on in March 1976 by paediatricians, surgeons, urologists and radiotherapists in Austria. Wilms' tumour was diagnosed in 34 children between 1 january 1976 an 29 february 1980 (stage I: n = 11, stage II: n = 8, stage III: n = 8, stage IV: n = 7). Parents of two children refused treatments; both children have since died of metastases. Of the remaining 32 children 29 (90.6%) are alive, 10 for more than 4, 15 for more than 3 and 19 for more than 2 years after diagnosis. 21 children are without need of treatment. Three children have died, one due to postoperative complications, one due to haemorrhagic chickenpox, but free of tumour, and one after insufficient treatment. Two of the five children with a recurrence between 2 1/4 to 15 months after diagnosis had been treated inadequately in the initial phase. The tumour free survival rate in 74.2%. Two children with early occurring or recurrent lung metastases have survived for 53 1/2 and 54 months up to now.

[Results of antireflux-surgery in children according to Leadbetter-Politano]. / Operationsergebnisse der Antirefluxplastik nach Leadbetter-Politano bei Kindern.

The indications for operation and results of antireflux-operation according to Leadbetter-Politano method are discussed in a retrospective evaluation of 80 children. Age of child, grade of reflux, cystoscopic findings of ureteral orifices and therapeutic success in treatment of urinary tract infection are the main criteria for the indication to surgical intervention. The operation was successful in 79 out of 80 children. Three of them needed a corrective operation. The number of postoperative complications was in accordance with results in the literature. The incidence of postoperative recurrence of urinary tract infection in 7.5% was relatively low.

[Long-term observations after adrenal surgery]. / Langzeitbeobachtung nach Nebennierenoperationen.

Long-term observations after adrenal surgery show different results according to indications for operations. The longest period of observation shows 8 patients with adrenal hyperplasia. In 3 of 4 bilaterally adrenalectomised patients no satisfactory results were obtained despite substitution treatment; 4 one-side adrenalectomised patients are in good condition. Operative results of 7 patients with benign phaeochromocytoma are satisfactory. Two patients with malignant phaeochromocytoma developed a local recidive and distant metastases. Carcinoma of the adrenal gland, if not hormonally active, were not diagnosed in early stage, 3 patients died from metastases within 3 years. A 2-years-old girl, who was operated on with hormonally active carcinoma, is in good condition 21 years later.

[Autologous ureter transplantation. Results of animal experiments]. / Autologe Uretertransplantation. Ergebnisse tierexperimenteller Untersuchungen

Autologous transplantation of the ureter in the whole lengths was carried out successfully. After unilateral nephrectomy the ureter was cut from the bladder, denudated and wrapped in the omentum majus. 4 weeks after that operation revascularisation of the ureter was pronounced so that the ureter could be used for reanastomosis with the renal pelvis and the bladder on the contralateral side. One dog lived with the transplanted ureter for one year and then died during birth of 5 puppies. One dog is living now since two years and is healthy.