J. P. Kaltwasser

Non‐invasive quantitation of liver iron‐overload by magnetic resonance imaging

A standard magnetic resonance imaging (MRI) system allowing spin echo times of 10 ms was used to quantitate liver iron concentration in nine healthy normal subjects and 13 patients with various grades of iron overload. Body iron status was estimated by measuring the serum ferritin concentration. In 11 subjects (two normal healthy controls, eight patients with HLA‐related hereditary haemochromatosis and one patient with thalassaemia major) non‐haem hepatic iron concentration was determined chemically in biopsy specimens (dry weight), in parallel to serum ferritin and MRI‐T2 relaxation times. A moderate correlation (r=0.79) was obtained for the correlation of the T2‐relaxation rate (1/T2) and serum ferritin of the 22 subjects investigated. A much closer correlation (r= 0.98) was observed for the 1/T2 liver iron relationship in the 10 subjects analysed by liver biopsy. It is concluded from these preliminary observations, that MR‐imaging may provide a useful non‐invasive tool for the quantitative determination of liver iron in iron overload‐syndromes.

Imbalance in distribution of functional autologous regulatory T cells in rheumatoid arthritis

Objectives: Regulatory T cells (Tregs) exert their anti-inflammatory activity predominantly by cell contact-dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA). Methods: 44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)γ-secreting cells was quantified in synovial tissue cell cultures, CD3-depleted synovial tissue cell cultures, synovial tissue cultures co-cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real-time PCR for CD3ε, T-bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein. Results: RA synovial tissue cell cultures exhibited spontaneous expression of IFNγ which was abrogated by depletion of CD3+ T cells and specifically reduced by co-culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T-bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity. Conclusion: This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments.

An endogenous retroviral long terminal repeat at the HLA-DQB1 gene locus confers susceptibility to rheumatoid arthritis

Human endogenous retrovirus (HERV) long terminal repeat (LTR) elements contain regulatory sequences that can influence the expression of adjacent cellular genes, which may contribute to breakdowns of the immune function leading to autoimmune disease. Rheumatoid arthritis (RA) is associated with particular HLA-DR/DQ haplotypes that modulate the pathogenesis of this autoimmune disease. We have therefore studied a solitary LTR element (DQ-LTR3) of the HERV-K family at the HLA-DQB1 locus for a possible disease association among 228 RA patients and 311 unrelated blood donors. The DQ-LTR3 was significantly more frequent among patients (76% vs 33%, OR = 5.07,p < 0.0001), with the majority of patients being heterozygous for the DQ-LTR3 (61% vs 22%, p < 0.0001). HLA-DRB1*04 positive patients did still differ for the presence of the DQ-LTR3 (88% vs 70%, OR = 3.03, p < 0.001), with an increase of both DQ-LTR3 homozygous and heterozygous patients, when compared to DRB1*04 positive controls (p = 0.0015). HLA-DR/DQ genotype analysis among HLA-DRB1*04 positive individuals revealed significantly more DQ-LTR3 homozygotes among HLA-DRB1*04-DQBI*03 homozygous patients (72% vs 27%, P = 0.015), and the number of DQ-LTR3 homozygous (23% vs 19%) and heterozygous (66% vs 53%) individuals was also increased among HLA-DRB1*04 heterozygous patients (p = 0.034). The presence of the DQ-LTR3 element increased both the relative risk and the positive predictive value for either DRB1*04-DQB1*03 positive/negative individuals when compared to the presence of HLA-DRB1*04-DQB1*03 alone. In conclusion, these data suggest that this DQ-LTR3 enhances susceptibility to RA.

Protection against severe disease is conferred by DERAA-bearing HLA-DRB1 alleles among HLA-DQ3 and HLA-DQ5 positive rheumatoid arthritis patients

Experimental studies in transgenic mice have suggested that HLA-DQ predisposes to rheumatoid arthritis (RA), but could also modulate disease severity by presenting peptides derived from self-DR molecules. In particular, a short amino acid sequence, (70)DERAA(74), in the third hypervariable region of HLA-DRB1 confers protection for the disease, while particular HLA-DQ [DQB1*0501/DQA1*01 (DQ5) and DQB1*03/DQA1*03 (DQ3)] molecules predispose to the disease. We have therefore analyzed the allelic distribution of HLA-DRB1, DQA1, and DQB1 and the presence of rheumatoid factor and nodules among 199 German RA patients and 196 healthy controls. Our results show that HLA-DQB1*03/DQA1*03 (or DRB1*04) predisposes to RA more than HLA-DQB1*0501/DQA1*01 (i.e., DRB1*01 and DRB1*10). Homozygosity for DQ3 confers the strongest genetic risk for RA (OR = 19.79 compared to OR = 10.05 for two doses of shared epitope (SE) positive HLA-DRB1 alleles). Furthermore, patients carrying both predisposing DQ and (70)DERAA(74)-positive HLA-DRB1 alleles are more often rheumatoid factor (RF) negative than patients carrying predisposing DQ alleles alone. Only one out of 14 patients (7%) with a protective combination (DQ3/(70)DERAA(74) and DQ5/(70)DERAA(74)) had rheumatoid nodules compared to 67 out of 144 patients (46.5%) with predisposing DQ alleles alone (OR = 0.12, 95% CI: 0.02-0.72, p = 0.004). These results demonstrate a protective role of (70)DERAA(74)-positive DRB1 alleles against disease severity among RA patients.

Total iron-binding capacity and serum transferrin determination under the influence of several clinical conditions.

In this study TIBC and serum-transferrin concentrations were determined by immunochemical turbidimetry, immunochemical nephelometry and radial immunodiffusion under normal and pathological clinical conditions. A total of 246 (123 male/123 female) patients were included [iron deficiency: 60 (18/42), iron overload: 56 (39/17), chronic inflammation: 47 (23/24), undefined diseases: 35 (16/19), healthy volunteers 48 (27/21)]. The data show that determination of TIBC from conversion of transferrin values using a constant factor results in significantly higher values compared to conversion with a function of first degree. For clinical practice the influence of different diseases is negligible. This study indicates that it is not possible to develop a universal algorithm for the conversion of transferrin values into TIBC.

Observational study of a patient and doctor directed pre-referral questionnaire for an early arthritis clinic

We evaluated a combined physician and patient questionnaire designed for identifying early rheumatoid arthritis (RA) and spondyloarthritis (SpA) in a cohort of 220 patients supposed for admission to an early arthritis clinic (EAC). The documents including personal and basis demographic data, referral diagnosis, questions related to RA and SpA classification criteria, functional limitations and previous diagnostic and therapeutic attempts were fax-transmitted to referring practices and returned before first EAC appointment. 125 referrals before introduction of the questionnaire served as controls. We found that a functional impairment of the hands provided more accurate prediction of RA than reports on morning stiffness or joint swelling. No clinical data proved predictive for SpA. We observed an unintended increase in the prescription of analgesics/NSAID and corticosteroids. In conclusion, questionnaires as designed here may provide substantial information for diagnosis of RA, but also imply the risk of unmeant therapeutic attempts.

A case of non-HFE juvenile haemochromatosis presenting with adrenocortical insufficiency [4]

The hepatitis C virus (HCV) has recently been implicated in the pathogenesis of several B-cell lymphoproliferative disorders. In particular, anti-HCV antibodies and/or HCV RNA have been detected in most patients with type II mixed cryoglobulinaemia and in a large proportion of Italian patients with B-cell non-Hodgkins lymphoma (NHL) (reviewed by Silvestri et al, 1996; Agnello, 1997; Ivanovski et al, 1998). Moreover, an asymptomatic monoclonal B-cell expansion has been demonstrated by molecular methods in < 20% of Italian patients with HCV-associated chronic liver disease (Franzin et al, 1995). The high prevalence of chronic HCV infection among patients with B-cell NHL has been observed primarily in Mediterranean countries, particularly in Italy (9±32%), where the prevalence of HCV carriers in the general population is quite high and ranges from 1% to 5 ́4%. A recent US study investigating predominantly Hispanic patients also showed a significantly higher prevalence of HCV infection in NHL patients (22%) than in age-matched controls (Zuckerman et al, 1997). However, other studies from the USA, Canada, Germany, the UK and France have not confirmed this association (reviewed by Germanidis et al, 1999). A possible explanation for this difference is the significantly lower prevalence of HCV infection in the general population of the latter countries. To test this last possibility, we investigated the prevalence of HCV infection in B-cell NHL patients from the Republic of Macedonia, which is characterized by a relatively high prevalence of HCV carriers within the general population (2 ́0%). We tested 112 consecutive patients with NHL (93 Macedonian and 19 Albanian) and a control group of 137 patients with other B-cell malignancies (38 with Hodgkins disease, 43 with chronic lymphocytic leukaemia, nine with acute lymphoblastic leukaemia, 26 with multiple myeloma and one with WaldenstroÈms macroglobulinaemia). The diagnosis was based on histological and immunohistochemical analysis of a lymph node and/or bone marrow biopsy according to the Revised European±American Classification (REAL) of lymphoid neoplasms. The serum samples were tested for antibodies to HCV by third-generation enzymelinked immunosorbent assay (ELISA) using commercially available kits and for HCV RNA by reverse transcriptase± polymerase chain reaction (RT±PCR) amplification of the 5 0 untranslated region of HCV. Table I summarizes the results for the different NHL subtypes and the controls. HCV infection was detected in only one patient with NHL (0 ́89%) and in one of the 137 patients with other B-cell malignancies (0 ́72%). Thus, our study demonstrates a low prevalence of HCV infection in patients with B-cell NHL from Macedonia and a lack of association between the two disorders. The comparable prevalence of HCV infection in the general population of Macedonia with that in Italy indicates that HCV infection requires additional environmental factors and/or a distinct genetic background to induce a malignant B-cell disorder. The same appears to be true for the benign HCV-associated B-cell lymphoproliferations, such as type II mixed cryoglobulinaemia, and the asymptomatic monoclonal B-cell expansions; both disorders were recently found to be significantly less prevalent in Japanese than in Italian patients with HCV-positive chronic liver disease (Pozzato et al, 1999). Therefore, it seems likely that HCV-associated lymphoproliferative disorders have a multifactorial aetiology, which includes additional, presently undetermined, genetic and environmental factors that may vary widely with geography.

Die hämochromatotische Arthropathie – Eine frühe Manifestation genetischer Hämochromatose

Summary Recent studies have shown a high frequency of genetic hemochromatosis in the Caucasion population. In addition, the well known organ involvement of genetic hemochromatosis was evident; more than 50% of patients develop a typical arthropathy which may result in severe physical disability. Among approximately 5000 patients referred to the rheumatology outpatient clinics of Bad Nauheim and Frankfurt with different rheumatologic diagnoses, 11 patients with typical signs of hemochromatotic arthropathy were identified. In none of those cases had the diagnosis “genetic hemochromatosis” been previously established. These patients had been treated for rheumatoid arthritis and other rheumatologic disorders over several years. All showed severe organ dysfunction due to iron overload, resulting in a reduced life expectancy. This investigation shows that knowledge of the typical signs of hemochromatotic arthropathy could lead to an earlier diagnosis of genetic hemochromatosis which is necessary to prevent the complications of iron overload in those patients.Zusammenfassung Nach neuen epidemiologischen Untersuchungsergebnissen ist die genetische Hämochromatose eine der häufigsten genetisch determinierten Stoffwechselerkrankungen in der kaukasischen Population. Die lebenslang währende Eisenüberladung führt bei mehr als der Hälfte der Patienten zu einer teilweise schweren Arthropathie. Die hämochromatotische Arthropathie wird häufig als Arthrose oder Rheumatoide Arthritis fehlgedeutet, obwohl sie einige recht typische Besonderheiten aufweist. Bei ca. 5000 Patienten, die in die Rheumasprechstunden der Kliniken Bad Nauheim und Frankfurt wegen unterschiedlichster rheumatologischer Diagnosen überwiesen wurden, konnten bislang 11 Patienten mit manifester Hämochromatose aufgrund des typischen Gelenkbefalls entdeckt werden. In allen Fällen fanden sich bereits ausgeprägte Organschädigungen, so daß die Lebenserwartung dieser Patienten eingeschränkt ist. Diese Untersuchung zeigt, daß die genetische Hämochromatose eine häufig übersehene Erkrankung ist. Die Kenntnis des Befallsmusters kann zur frühen Diagnose einer genetischen Hämochromatose führen und somit Organschäden vermeiden.

Prednisolone induces interleukin-18 expression in mononuclear blood and myeloid progenitor cells

Abstract.Objective: Interleukin (IL)-18 is involved in host defense mechanisms and inflammatory diseases, among them rheumatoid arthritis (RA). High levels of IL-18 expression in RA joints are contrasted by reduced IL-18 expression in RA peripheral blood mononuclear cells (PBMC). Here, we investigated a putative IL-18 regulating role of corticosteroids.¶Methods: IL-18 transcript and protein levels in PBMC from untreated and prednisolone treated RA patients, and from healthy donors were assessed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblotting. IL-18 regulation was determined in PBMC and U937 cells upon exposure to prednisolone in vitro by RT-PCR and Northern Blot analysis, by ELISA in cell culture supernatants, and in transiently transfected THP-1 cells by IL-18 promoter activity luciferase assays.¶Results: In RA PBMC, IL-18 transcript levels were dose dependently restored, in parallel with administered prednisolone treatment, to subnormal levels. The corresponding intracellular IL-18 deposits in contrast were depleted. In cultured PBMC and promonocytic cell lines, prednisolone up-regulated IL-18 transcription in parallel with increasing the IL-18 protein release into cell culture supernatants.¶Conclusion: Prednisolone increases IL-18 expression and release in PBMC and monocytic cell lines.

Severe juvenile haemochromatosis (JH) miassing HFE gene variants: implications for a second gene locus leading to iron overload

Juvenile haemochromatosis (JH) is a rare genetic disorder characterized by severe iron overload syndrome with clinical manifestation below the age of 30 years (Kaltwasser. 1998). The clinical course is characterized by heart failure and hypogonadotropic hypogonadism (Cazzola et al, 1983). It has been proposed that JH is an early-onset variant of genetic haemochromatosis (GH). In contrast to GH. a clear link of JH to the MHC locus on chromosome 6 is not found. and most patients are negative for HLA-A3 and B7. Gene variants of the HFEgene. a HLAclass I like gene on chromosome 6 have been implicated in the aetiology of iron overload in GH. Two mutations have been identified, one at position 282 leading to an exchange from cysteine to tyrosine (C282Y) and the second at position 63 leading to an exchange from histidine to glycine (H63D) (Feder ei al. 1996). In particular. homozygosity for the C282Y mutation is strongly associated with the presence of GH. We have previously studied HFE gene variants in German GH patients and their family