The mouse B cell repertoire : antibody specificities and immunoglobulin (sub) class distribution

The total number of different immunoglobulin (I g) molecules that the immune system produces is often called the antibody specificity repertoire orB cell repertoire (Chapter 1). This repertoire can be subdivided into three categories: the potential, the available and the actual repertoires. The potential repertoire is determined by the number, structure and mechanisms of expression of the germl ine genes encoding lg molecules plus the possible somatic variants derived from them and can be regarded as what potentially can be made. The available repertoire is defined as the set of diverse antibody molecules that are expressed by immunocompetent but resting B lymphocytes and can be looked upon as what has been made and can be used. The actual repertoire is represented by that set of antibodies that is actually secreted by S cells and can be regarded as what is actually being used. Little is known about the regulatory mechanisms that enable the establishment, from the potential repertoire, of the available and functionally expressed repertoire of the immunocompetent resting B cell compartment. Similarly, the mechanisms that govern the establishment of the actual repertoire from the available repertoire are only partly known. Therefore~ the purpose of the studies presented in this thesis (as outlined in Chapter Ill) was to obtain more information concerning the regulatory mechanisms that are involved in the functional expression of the lg C and V genes by murine B cells. To this end, frequency analyses of B cells secreting particular lg heavy chain isotypes (C gene expression) and specific lgM antibodies (V gene expression) were performed among the progeny of B cells that had differentiated from pre-S cells in vitro. The same analyses were performed on in vivo generated mitogen-reactive S cells (available repertoire) and on the 1Spontaneously1 occurring ( 1background') lg-secreting cells (actual repertoire). The possible regulating influences studied include age, T cells and exogenous antigens. The latter became feasible, since, with the successful breeding of germfree mice fed an ultrafiltered solution of chemically defined low molecular weight nutrients, exogenous stimuli such as antigens and mitogens can be reduced to a minimum never attained before

Molecular and flow cytometric analysis of the Vβ repertoire for clonality assessment in mature TCRαβ T-cell proliferations

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of Vbeta-Cbeta transcripts and flow cytometry with a Vbeta antibody panel covering more than 65% of Vbeta domains was validated using 28 SB-defined clonal T-cell receptor (TCR)alphabeta(+) T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V(beta) RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vbeta-Cbeta RT-PCR products were detected in all 47 samples, whereas single Vbeta domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vbeta monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vbeta gene segments not covered by the applied Vbeta monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vbeta-Cbeta products and single Vbeta domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vbeta analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vbeta antibody studies can be used as a powerful screening method for the detection of single Vbeta domain expression, followed by molecular studies in patients with more than 20% single Vbeta domain expression or large suspect T-cell populations (more than 50%-60%) without Vbeta reactivity

Paraneoplastic cerebellar degeneration associated with antineuronal antibodies: analysis of 50 patients

Paraneoplastic cerebellar degeneration (PCD) is a heterogeneous group of disorders characterized by subacute cerebellar ataxia, specific tumour types and (often) associated antineuronal antibodies. Nine specific antineuronal antibodies are associated with PCD. We examined the relative frequency of the antineuronal antibodies associated with PCD and compared the neurological symptoms and signs, associated tumours, disability and survival between groups of PCD with different antibodies. Also, we attempted to identify patient-, tumour- and treatment-related characteristics associated with functional outcome and survival. In a 12-year period, we examined >5000 samples for the presence of antineuronal antibodies. A total of 137 patients were identified with a paraneoplastic neurological syndrome and high titre (> or =400) antineuronal antibodies. Fifty (36%) of these patients had antibody-associated PCD, including 19 anti-Yo, 16 anti-Hu, seven anti-Tr, six anti-Ri and two anti-mGluR1. Because of the low number, the anti-mGluR1 patients were excluded from the statistical analysis. While 100% of patients with anti-Yo, anti-Tr and anti-mGluR1 antibodies suffered PCD, 86% of anti-Ri and only 18% of anti-Hu patients had PCD. All patients presented with subacute cerebellar ataxia progressive over weeks to months and stabilized within 6 months. The majority of patients in all antibody groups had both truncal and appendicular ataxia. The frequency of nystagmus and dysarthria was lower in anti-Ri patients (33 and 0%). Later in the course of the disease, involvement of non-cerebellar structures occurred most frequently in anti-Hu patients (94%). In 42 patients (84%), a tumour was detected. The most commonly associated tumours were gynaecological and breast cancer (anti-Yo and anti-Ri), lung cancer (anti-Hu) and Hodgkin's lymphoma (anti-Tr and anti-mGluR1). In one anti-Hu patient, a suspect lung lesion on CT scan disappeared while the PCD evolved. Seven patients improved by at least 1 point on the Rankin scale, while 16 remained stable and 27 deteriorated. All seven patients that improved received antitumour treatment for their underlying cancer, resulting in complete remission. The functional outcome was best in the anti-Ri patients, with three out of six improving neurologically and five were able to walk at the time of last follow-up or death. Only four out of 19 anti-Yo and four out of 16 anti-Hu patients remained ambulatory. Also, survival from time of diagnosis was significantly worse in the anti-Yo (median 13 months) and anti-Hu (median 7 months) patients compared with anti-Tr (median >113 months) and anti-Ri (median >69 months). Patients receiving antitumour treatment (with or without immunosuppressive therapy) lived significantly longer [hazard ratio (HR) 0.3; 95% confidence interval (CI) 0.1-0.6; P = 0.004]. Patients > or =60 years old lived somewhat shorter from time of diagnosis, although statistically not significant (HR 2.9; CI 1.0-8.5; P = 0.06)

<i>Campylobacter jejuni </i>infections and anti-GM1 antibodies in Guillain-Barré syndrome

The group of patients with Guillain-Barre syndrome (GBS) is very heterogenous with regard to antecedent infections, immunological parameters, clinical manifestations, and response to treatment. In this study, the presumed pathogenic factors anti-GM1 antibodies and Campylobacter jejuni infections were related to the clinical characteristics. Serum from 154 patients with GBS, 63 patients with other neurological diseases (OND), and 50 normal controls (NC) were tested for the presence of antibodies against GM1 and C. jejuni. Anti-GM1 antibodies were detected in 31 (20%) GBS patients, 5 (8%) OND patients, and in none of the NC. Evidence for a recent C. jejuni infection was found in 49 (32%) GBS patients and less often in OND patients (11%) or NC (8%). In GBS patients, the presence of anti-GM1 antibodies was significantly associated with C. jejuni infections. The subgroup of GBS patients with anti-GM1 antibodies suffered more often from a rapidly progressive and more severe neuropathy with predominandy distal distribution of weakness, without deficits of cranial nerves or sensory disturbances. The subgroup with C. jejuni infection also more often had a severe pure motor variant of GBS. Recovery of the patients with anti-GMl antibodies and C. jejuni infections was not as good after plasma exchange compared with intravenous immunoglobulins.</p

The identification of celiac disease in asymptomatic children: the Generation R Study

Background: The objective of our study was to assess whether TG2A levels in the healthy childhood population can be predictive of subclinical CD. Methods: A total of 4442 children (median age, 6.0 years) participating in a population-based prospective cohort study were screened on serum TG2A. Those with positive TG2A (≥7 U/ml; n = 60, 1.4%) were invited for clinical evaluation (median age, 9.0 years). Medical history, physical examination, serum TG2A, and IgA-endomysium (EMA) were assessed, as well as HLA DQ 2.2/2.5/8 typing. Patients with positive serologies and genetic risk types underwent duodenal biopsies. TG2A levels at the time of biopsy were compared with the degree of enteropathy. Results: Fifty-one TG2A-positive children were included in the follow-up: 31 (60.8%) children had CD, ten (19.6%) did not have CD, and ten (19.6%) were considered potential CD cases because of inconclusive serologies. Duodenal biopsies were performed in 26/31 children. CD with Marsh 3a/b enteropathy was observed in 75% (15/20) of children having TG2A levels ≥10ULN at 6 years of age, as well as in 75% (6/8) of children having a positive TG2A <10 ULN (OR 1.00; 95% CI 0.15–6.64). CD cases had a lower BMI SDS (mean −0.49, SD 0.92) than children without CD (mean 0.47, SD 1.37; p = 0.02). No differences were observed in gastrointestinal symptoms. Conclusions: Serum TG2A screening at 6 years of age in the healthy childhood population has a positive predictive value of 61% to detect subclinical CD. We did not find a positive correlation between serum TG2A levels and the degree of enteropathy