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We have analyzed gallstones from four patients of Europe and particularly from England (including samples from a mother and a daughter) and Greece. According to the XRD, FTIR, NMR and laser micro-Raman results the studied materials correspond to typical cholesterol monohydrate (ChM). The micro-morphology of cholesterol microcrystals was investigated by means of SEM-EDS. The XRF results revealed that Ca is the dominant non-organic metal in all gallstones (up to ∼1.95wt.%) together with Fe, Cu, Pb and Ni (up to ~19ppm for each metal). Gallstones from England contain additional Mn (up to ~87ppm) and Zn (up to ∼6ppm) while the sample of the mother contains negligible Zn and Mn, compared to that of her daughter, but significant As (~4.5ppm). All cholesterol gallstones examined are well enriched in potentially toxic metals (Pb, as well as Ni in one case) and metalloids (As also in one case) as compared to the global average. The position of Zn, which is a characteristic biometal, in the structure of cholesterol, was investigated by molecular simulation using the Accelrys Materials Studio(®) software. On the basis of IRMS results, all gallstones examined exhibit a very light δ(13)C signature (average δ(13)C ~-24‰ PDB). Gamma-ray spectrometry measurements indicate the presence of (214)Pb and (214)Bi natural radionuclides due to the (238)U series as well as an additional amount of (40)K., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Introduction: This study investigated five confirmed rheumatoid arthritis (RA) susceptibility genes/loci (HLA-DRB1, PTPN22, STAT4, OLIG3/TNFAIP3 and TRAF1/C5) for association with susceptibility and severity in an inception cohort., Methods: The magnitude of association for each genotype was assessed in 1,046 RA subjects from the Yorkshire Early RA cohort and in 5,968 healthy UK controls. Additional exploratory subanalyses were undertaken in subgroups defined by autoantibody status (rheumatoid factor and anti-cyclic citrullinated peptide) or disease severity (baseline articular erosions, Health Assessment Questionnaire (HAQ) score and swollen joint count (SJC))., Results: In the total RA inception cohort, the HLA-DRB1 shared epitope (per-allele odds ratio (OR) = 2.1, trend P < 0.0001), PTPN22 (per-allele OR = 1.5, trend P < 0.0001), OLIG3/TNFAIP3 locus (per-allele OR = 1.2, trend P = 0.009) and TRAF1/C5 locus (per-allele OR = 1.1, trend P = 0.04) were associated with RA. The magnitude of association for these loci was increased in those patients who were autoantibody-positive. PTPN22 was associated with autoantibody-negative RA (per-allele OR = 1.3, trend P = 0.04). There was no evidence of association between these five genetic loci and baseline erosions or SJC in the total RA cohort, after adjustment for symptom duration. TRAF1/C5 was significantly associated with baseline HAQ, however, following adjustment for symptom duration (P trend = 0.03)., Conclusions: These findings support the mounting evidence that different genetic loci are associated with autoantibody-positive and autoantibody-negative RA, possibly suggesting that many of the genes identified to date are associated with autoantibody production. Additional studies with a specific focus on autoantibody-negative RA will be needed to identify the genes predisposing to this RA subgroup. The TRAF1/C5 locus in particular warrants further investigation in RA as a potential disease severity locus.

Objective: To characterise and investigate the functional consequences of a novel TNFRSF1A splice site mutation causing tumour necrosis factor receptor associated periodic syndrome (TRAPS) in a 16-year-old male patient and his mother., Methods: Mutational DNA screening was performed in the patient and his mother. Western blotting was used to analyse protein expression levels of TNFR1. A multiplex bead immunoassay was used to quantify serum levels of range of cytokines, and an ELISA-based transcription factor assay to measure nuclear factor (NF)-kappaB transactivation. Serum levels of soluble TNFR1 (sTNFR1) were measured by ELISA and fluorescence-activated cell sorting (FACS) analysis used to measure monocyte TNFR1 cell surface expression., Results: A novel mutation, c.472+1G>A (C158delinsYERSSPEAKPSPHPRG), involving a splice site in intron 4 of TNFRSF1A, was found in the proband and affected mother leading to a 45 nucleotide insertion of intronic DNA into the mRNA, resulting in an in-frame insertion of 15 amino acids in the mature TNFR1 protein and a deletion of a cysteine residue C129 (158) in cysteine rich domain (CRD)3. The patients had reduced serum sTNFR1 and surface expression levels of TNFR1, with marked increases in pro- and anti-inflammatory cytokine. Their peripheral blood mononuclear cells (PBMC) had increased basal NF-kappaB activation compared with healthy controls and also had increased p50 nuclear expression following tumour necrosis factor (TNF) stimulation compared with PBMC from healthy controls, as well as T50M (T79M) and C88R (C117R) patients with TRAPS and patients with rheumatoid arthritis (RA)., Conclusion: A novel, TRAPS causing, TNFRSF1A splice site mutation is associated with decreased sTNFR1 levels, cell surface and whole cell extract expression and increased NF-kappaB transcription factor activation.

Objective: Many classification systems for the HLA-DRB1 allelic association with rheumatoid arthritis (RA) have been reported, but few have been validated in additional populations. We sought to evaluate 3 different DRB1 allele classification systems in a large cohort of Caucasian RA patients and control subjects in the UK., Methods: HLA-DRB1 typing was undertaken in 1,325 Caucasian RA patients and 462 healthy Caucasian controls who were residents of the UK. Logistic regression analyses were performed to investigate the different classification systems., Results: We confirmed the association between the susceptibility alleles S2 and S3P, as proposed by Tezenas du Montcel, and the presence of RA in UK Caucasians. A significant hierarchy of risk was observed within the S3P allele group. There was no evidence of a significant association between DRB1*1001 and RA. Our data did not support the hypothesis that an isoleucine at position 67 conferred protection against RA, other than in contrast to the susceptibility alleles. However, the presence of an aspartic acid at amino acid 70 did appear to confer some degree of protection., Conclusion: We were unable to fully substantiate any of the 3 recent revisions of the shared epitope hypothesis in this large cohort of Caucasian RA patients and control subjects in the UK. This reinforces the importance of evaluating disease susceptibility alleles in different Caucasian populations as well as in other ethnic groups. In particular, it will be important to clarify the precise DRB1 association in a given population before DRB1 genotyping is incorporated into clinical diagnostic or treatment algorithms.

Background: Immunological tolerance in humans using anti-T-cell monoclonal antibodies (mAbs) may be hampered by a pro-inflammatory microenvironment. All clinical trials of such therapies in rheumatoid arthritis (RA), however, have selected patients with active disease at baseline. Concurrent neutralization of inflammation with a TNFalpha antagonist should maximize the potential of anti-T-cell mAbs to induce tolerance in RA., Aim: To evaluate the safety of combining a TNFalpha antagonist and CD4 mAb in RA., Design: An iterative pilot study focused on the safety of such combination therapy., Methods: Eight poor prognosis, seropositive RA patients were treated with combined CD4 and TNFalpha blockade. Prolonged CD4 blockade was achieved with a humanized mAb, and TNFalpha blockade with a p55 TNF receptor fusion protein., Results: There was a low incidence of classical first-dose reactions to the CD4 mAb, possibly reflecting concomitant TNFalpha blockade. An unusual anaphylactoid reaction was seen, however, and one patient developed a probable allergic reaction after several infusions. Skin rashes were common, as previously reported with CD4 mAb monotherapy. No serious infections were documented during follow-up, despite CD4+ lymphopenia in some patients. Most patients appeared to demonstrate improved RA disease control after the study. After 17-49 months after therapy, one patient was in remission, one remained off disease modifying anti-rheumatic drugs and five had stable disease, three on previously ineffective doses of methotrexate., Conclusion: We report, for the first time in man, immunotherapy with a combination of an anti-cytokine and an anti-T-cell reagent. We witnessed an unusual first-dose reaction but there were no significant infectious complications.

Objectives: This study investigated the clinical impression that there was an increased prevalence of respiratory disorders in both the Hypermobility Syndrome (HMS)/Benign Joint Hypermobility Syndrome (BJHS) and Ehlers-Danlos Syndrome (EDS), compared with the normal population., Methods: A questionnaire was distributed to 509 subjects (221 healthy controls, 126 HMS, 162 EDS) who documented respiratory symptoms and previously diagnosed respiratory and atopic disorders. A subgroup of 157 responders underwent full clinical and serological assessments, and 57 subjects were assessed physiologically., Results: A significant increase in the frequency of a wide range of respiratory symptoms and reduced exercise tolerance was observed in subjects with both HMS and EDS compared with controls. In particular, there was an increased prevalence of asthmatic symptoms (HMS: OR 2.4, 95% CI 1.4-4.1, p = 0.002; EDS: OR 3.1, 95% CI 1.8-5.2, p<0.001) and atopy (HMS: OR 2.7, 95% CI 1.6-4.5, p<0.001; EDS: OR 2.6, 95% CI 1.6-4.4, p<0.001), which was subsequently confirmed by clinical assessment. Pulmonary physiological studies revealed increased lung volumes, impaired gas exchange and an increased tendency of both the lower and upper airways to collapse., Conclusions: We have demonstrated, for the first time, that individuals with HMS/BJHS and EDS have respiratory symptoms in association with various pulmonary physiological abnormalities. The increased prevalence of asthma may be due to linkage disequilibrium between the genes causing these conditions or a function of the connective tissue defect itself. In the non-asthmatic population, changes in the mechanical properties of the bronchial airways and lung parenchyma may underlie the observed increased tendency of the airways to collapse.

The aim of this study was to relate serum immunoglobulin G2 subclass levels in a large paediatric population with cystic fibrosis, to clinical status and antibody levels to Haemophilus influenzae type b and Streptococcus pneumoniae and to observe any changes over a 2-year period. IgG subclasses were measured in 131 patients. Results were compared with levels from age-related normal population data. The following clinical data were collected at baseline and 2 years later; genotype: height, weight, and BMI z-scores: FEV1 (as percent predicted): Shwachman-Kulczcyki and Northern chest X-ray scores: Pseudomonas aeruginosa status. Antibody levels to H. influenzae type b and S. pneumoniae measured at baseline were related to IgG2 level. There was a reduction in the prevalence of low levels of IgG2 from 29% to 10% over the 2-year period. Low levels of IgG2 were not associated with any decline in clinical well-being. Low levels of IgG2 alone were associated with low antibody levels to S. pneumoniae. Low levels of IgG2 and low levels of antibody to H. influenzae and S. pneumoniae were not associated with any decline in clinical well-being. Children with high levels of IgG2 had worse lung function, worse Shwachman-Kulczcyki and Northern chest X-ray scores and higher levels of P. aeruginosa infection. Children with low IgG2 levels were not worse clinically compared to those with normal or high IgG2 levels. High IgG2 levels were associated with a worse clinical status., ((c) 2006 Wiley-Liss, Inc.)

The aim of this study was to report serum immunoglobulin (Ig) and IgG subclass levels in a large pediatric population with cystic fibrosis, and relate these to measures of disease severity. Total immunoglobulin levels were measured in 154 patients, and IgG subclass levels were measured in 136 patients and compared to age-related normal population data and to levels reported in previously published studies of children with cystic fibrosis. Clinical data were also collected: genotype; height, weight, and BMI standard deviation scores; FEV(1) (as percent predicted); Shwachmann-Kulczycki (S-K) and Northern chest X-ray scores; and Pseudomonas aeruginosa infection status. The clinical well-being of patients with hypo- or hyper-gammaglobulinemia was compared with age- and sex-matched control patients who had normal levels of gammaglobulin. IgG subclass levels were measured, and the results were compared with previous studies. Eleven patients had hypergammaglobulinemia (7.8% compared with 0-69% in the published literature). Patients with hypergammaglobulinemia had lower FEV(1) percent-predicted values, and worse S-K and Northern chest X-ray scores than controls. Three patients had hypogammaglobulinemia (1.9% compared with 0-10.8% in the published literature). There was no difference in any clinical parameter between controls and those with hypogammaglobulinemia. Nineteen patients (14%) had low levels of IgG1, and 40 patients (29%) had low levels of IgG2. The low percentage of patients with abnormally high immunoglobulin levels probably reflects the improved respiratory status of today's children with CF. The low percentage of those with low IgG probably reflects better nutritional status. The finding of worse lung function and clinical scores in patients with hypergammaglobulinemia agrees with the published literature. The high percentage of patients with low IgG2 was unexpected and was not previously reported. The clinical significance of this in patients with CF is unknown.

Aims: To determine whether polymorphisms of the genes encoding donor or recipient interleukin 1alpha (IL-1alpha), tumour necrosis factor alpha (TNFalpha), or IL-4 have any impact on the incidence of acute rejection after renal transplantation., Methods: All donors and recipients were genotyped for three polymorphisms in the three cytokine genes: IL1A -889, TNFA -308, and IL4 -590., Results: Statistical analysis of the data obtained revealed no association between the cytokine gene polymorphisms tested and the incidence of post-transplant acute rejection. After stratification for human leucocyte antigen (HLA) matching, it was found that kidneys from donors positive for the TNFA-A allele had a significantly increased incidence of acute rejection in HLA-DR mismatched transplants., Conclusions: This finding argues for prospective TNFA genotyping of renal donors, with avoidance of allocation of kidneys from donors positive for the TNFA-A allele to HLA-DR mismatched recipients.

Background: Use of intravenous (IV) immunoglobulin (Ig) to obtain panel reactive antibody (PRA) A reduction in sensitized patients has been widely reported. Because no IVIg preparation is formulated specifically for this purpose, the authors have sought to determine whether, through laboratory testing, they could guide the rational choice of product for clinical use., Methods: Using a flow cytometric approach, the authors have quantitatively determined the capacity of 22 different IVIg preparations to cause PRA reduction., Results: IVIg preparations showed considerable variability in their individual capacity to reduce serum PRA. Protein-A pretreatment of IVIg preparations was found to reduce their capacity to cause PRA reduction., Conclusion: Laboratory screening of IVIg preparations provides a rational basis for the selection of product for administration to patients in whom the aim is to produce a PRA reduction. Experiments involving protein-A treatment of IVIg preparations indicate that immunoglobulin G is the principal factor involved in the abrogation of serum reactivity.

Purpose: To investigate the role of the shared epitope alleles in determining susceptibility to and the phenotype of corneal melting in patients with rheumatoid arthritis (RA)., Methods: The HLA class 1 and 2 genotype was determined for 17 patients with rheumatoid-associated comeal melting by the phototyping method. HLA-DR4 subtyping was performed by PCR sequence-based typing. The frequency of all the shared epitope alleles and, in particular, of the higher-risk *0401 and *0404 alleles, was compared with healthy controls and unrelated RA patients, with and without extra-articular manifestations. A comparison was also made between the shared epitope genotype of the corneal melt patients and local, ocular disease characteristics., Results: Thirteen (76%) patients with corneal melt possessed at least one shared epitope allele and 5 (29%) possessed two alleles. The dominant alleles were variants of the DR4 family, notably the *0401, *0404 and *0408 alleles. Both the allele frequency and a double dose of shared epitope alleles were more common in the three RA patient groups than in the healthy, control group (p < 0.005). Although the frequency of the higher-risk alleles was similar in the three RA patient group, a trend existed for a double dose of higher-risk alleles to be more common in the patients with either corneal melt or other extra-articular manifestations (p > 0.2). No association was found between the number or type of shared epitope alleles and any of the ocular disease characteristics studied., Conclusions: The results of this study suggest that the shared epitope alleles do not influence the ocular disease phenotype of corneal melt in RA patients. Shared epitope determination of RA patients may help to identify those susceptible to either corneal melt or other extra-articular disease. RA patients with a double dose of higher-risk alleles may have an increased risk of corneal melt.

Problem: Recurrent pregnancy loss (RPL) affects 2-4% of couples, and remains largely unexplained. Recent studies have examined the role of cytokines in the maintenance of normal pregnancy, which is linked with an increased expression of Th2 cytokines. Overexpression of Th1 cytokines is associated with RPL. Knowing that functional polymorphisms exist for certain cytokines, it has therefore been suggested that women with RPL may have a genetic predisposition to overexpress Th1 cytokines., Method of Study: The genes for interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) carry functional gene polymorphisms. In both cases these are biallelic polymorphisms that can be detected by polymerase chain reaction followed by restriction fragment length polymorphism. The aim of this pilot study was to assess whether carriage of the rarer alleles (TNF*2 and IL-1B*2) could act as independent risk factors in recurrent miscarriage., Results: We found an increased incidence in the carriage of TNF*2, more pronounced in those women with two or more miscarriages. Carriage of the IL-1B*2 either alone or in association with TNF*2 was not associated with recurrent miscarriage., Conclusion: There may be a role for these cytokine gene polymorphisms in RPL.

Objective: To investigate a possible association between a functional polymorphism in the intermediate-affinity receptor for IgG called Fc-gamma receptor type IIIA (FcgammaRIIIA [CD16]) and rheumatoid arthritis (RA)., Methods: This was an allelic association study in which a single nucleotide polymorphism in FcgammaRIIIA was examined as a susceptibility and/or severity factor for RA. The FcgammaRIIIA-158V/F polymorphism was genotyped by direct sequencing in 2 well-characterized ethnic groups, UK Caucasians (141 RA patients and 124 controls) and North Indians and Pakistanis (108 RA patients and 113 controls)., Results: The FcgammaRIIIA-158V/F polymorphism was associated with RA in both ethnic groups (P = 0.028 for UK Caucasians, P = 0.050 for North Indians and Pakistanis, and P = 0.003 for both groups combined). FcgammaRIIIA-158VF and -158W individuals had an increased risk of developing RA in both populations (UK Caucasians odds ratio [OR] 1.6, P = 0.050; North Indians and Pakistanis OR 1.9, P = 0.023; and combined groups OR 1.7, P = 0.003). In the UK Caucasian group, the highest risk was for nodular RA, a more severe disease subset, associated with homozygosity for the FcgammaRIIIA-158V allele (OR 4.4, P = 0.004). There was also evidence for an interaction between the RA-associated HLA-DRB1 allele and the presence of at least 1 FcgammaRIIIA-158V allele in predicting susceptibility to RA (OR 5.5, P = 0.000)., Conclusion: We have demonstrated that the FcgammaRIIIA-158V/F polymorphism is a susceptibility and/or severity marker for RA in 2 distinct ethnic groups. This finding may ultimately provide additional insights into the pathogenesis of RA and other autoantibody/immune complex-driven autoimmune diseases.

Aims: To investigate polymorphism within the tumour necrosis factor alpha (TNF-alpha) promoter region and within the interleukin-1 receptor antagonist (IL-1Ra) gene in a group of patients with vasculitis associated corneal melting., Methods: The polymorphic regions at position -308 on the TNF-alpha promoter region and in intron 2 of the IL-1Ra gene were amplified by the polymerase chain reaction (PCR). The resultant products were separated by electrophoresis on agarose gels and visualised by ethidium bromide staining. Genotype and allele frequencies for the 20 patients were compared with healthy controls from the same geographical area., Results: The allele frequencies in the patient and control groups respectively for the TNF-alpha and IL-1Ra sites studied were as follows: TNF1, 82.5% and 80.2%; TNF2, 17.5% and 19.8%; IL-1Ra*1, 82. 5% and 78.3%; IL-1Ra*2, 15% and 20%; IL-1Ra*3 2.5% and 1.5%. Although there was a trend for the IL1Ra*2 allele to be more common in the control group, no allele was found to have a statistically significantly association with the patient group: TNF1 p = 0.89; TNF2 p = 0.89; IL-1Ra*1 p = 0.65; IL-1Ra*2 p = 0.68; IL-1Ra*3 p= 0. 50., Conclusions: The results suggest that the polymorphic alleles of TNF-alpha and IL-1Ra studied play little or no part in the susceptibility to corneal melting among these patients with systemic vasculitis.

The production of superoxide by the peripheral blood neutrophils of 19 patients with active rheumatoid arthritis was measured during treatment with sulphasalazine (SASP). The response to drug treatment was determined by change in plasma viscosity, CRP, early morning stiffness and articular index over a 10-point scale. Of the 19 patients studied, eight were considered to have responded well to SASP and seven to have responded poorly or not at all. Over the treatment period, plateau levels of superoxide production fell in seven of the eight responders (P = 0.028) compared with a non-significant fall in 3/7 of the non-responder groups. The initial rate of superoxide production also fell in the responder group, but this was not statistically significant. Initial values in both the responder and non-responder groups were comparable with those seen for normal controls. Analysis of drug levels showed all patients to be compliant with drug treatment; however, drug levels and neutrophil activity were not correlated. Studies of the effect of SASP and sulphapyridine on superoxide production in vitro showed no difference between good and poor responders. These results suggest that there is no inherent difference between good and poor responders regarding the susceptibility of their neutrophils to SASP. SASP's action on neutrophils, therefore, appears not to be its main mechanism of disease-modifying activity in RA.

Aim-To determine the relation of the low anticoagulant response phenotype with the Factor V Q506 (Leiden) mutation in a cohort of patients with thrombophilia.Methods-Fifty four patients with either a personal or family history of deep vein thrombosis were investigated both for their anticoagulant response by the activated protein C resistance test (APCR) and their genetic status in respect of the Leiden mutation by means of a PCR-RFLP method.Results-Low APCR ratios do not necessarily predict possession of the Leiden mutation. Conversely, normal ratios do not exclude the mutation. Of 14 individuals with low APCR ratios, the Leiden mutation was absent in five. Of the remainder, three were heterozygous and six homozygous. Of nine heterozygote individuals, only three had low APCR ratios. All patients homozygous for the defect had low APCR ratios.Conclusions-These results lend further weight to the hypothesis that the APC resistant phenotype results from more than one genetic defect and indicate the value of combined functional and molecular investigations in all patients with thrombophilia.

We have reviewed 39 adult patients who presented over a 5-year period with biopsy confirmed renal disease in association with positive antineutrophil cytoplasmic antibody (18 with C-ANCA, 21 with P-ANCA). Twenty-three (59%) had primary systemic vasculitis, typically with aggressive renal histology including focal necrotising and crescentic glomerulonephritis. In the remaining patients a wide range of clinical syndromes and renal histological appearances were apparent: 30 had abnormal renal function (serum creatinine > 140 mumol/l), including 17 who were initially dialysis-dependent. Intensive immunosuppression was administered in 33 cases. When response was assessed at 3 months, renal function was stable or improved in 17 (52%), 5 of whom were able to discontinue dialysis. There was, however, an appreciable early mortality and, at latest follow-up (1-57 months), 12 patients had died and 8 were on the dialysis programme. On immunosuppression, ANCA became negative in the majority (median time 1.5 months) but recurred during clinical relapse in 11 cases. In asymptomatic patients (12 cases), the reappearance of ANCA positivity did not herald clinical relapse. The ANCA assay has increased awareness of systemic vasculitis but not removed the need for histological confirmation before instituting immunosuppression.

Faecal and serum samples were collected from 31 patients with active RA during treatment with DMARD sulphasalazine (SASP). These were examined for changes in faecal flora and antibodies to bacterial antigens respectively. Faecal counts of Clostridium perfrigens but not Escherichia coli or total aerobic or anaerobic counts fell significantly after 2 weeks of treatment, this decrease being maintained throughout the treatment period. There was, however, no relationship between changes in the faecal carriage of this micro-organism and response to drug treatment, as assessed using clinical and biochemical indicators of disease activity. Changes in antibody levels to antigen preparations of this organism were also unrelated to response to drug treatment. These results suggest that the anti-rheumatic properties of SASP are independent of its antibacterial effect on bacteria in the bowel and also that neither faecal carriage of, nor antibody responses to this bacterium are involved in disease pathogenesis. Antibody levels to an antigen preparation of Cl. perfringens were found to be significantly lower in those patients who respond well to SASP than those patients who show poor response; this may prove useful as a clinical marker for predicting those patients likely to respond to SASP therapy.

The 20-year-old girl we describe presented with myelofibrosis and systemic lupus erythematosus (SLE), which initially responded to treatment with corticosteroids but during a relapse 9 months later a bone marrow biopsy revealed no improvement in her myelofibrosis. As the cytotoxic treatments used to treat severe SLE may be associated with bone marrow suppression, it is important to consider the possibility of myelofibrosis complicating SLE, which, even when it is a presenting feature, may not readily respond to corticosteroids. When this girl was subsequently treated with high dose steroid and azathioprine her myelofibrosis went into remission.

The faecal flora and bacterial antibody levels of 22 patients with active rheumatoid arthritis (RA) were compared with those of 26 patients with osteoarthritis (OA) undergoing comparable treatment with non-steroidal anti-inflammatory drugs (NSAIDs), and a further 22 patients with OA who were not receiving NSAIDs. Faecal counts of Clostridium perfringens were significantly higher in the RA patient group and in those OA patients receiving NSAIDs, compared with those OA patients not taking NSAIDs (P = 0.032, P = 0.0004 respectively). Total aerobic and anaerobic counts were, however, identical in all three groups. Levels of serum IgA antibody to the alpha toxin of Cl. perfringens were higher in the RA group and in the OA group taking NSAIDs than in OA patients not taking NSAIDs (P = 0.011, P = 0.055). Serum IgG antibody to alpha toxin was higher in the RA group than in OA patients both on and off NSAIDs (P = 0.019, P = 0.0072) and also a group of normal controls (P = 0.032). These results suggest that the increased faecal counts of Cl. perfringens together with the associated increased antibody levels seen in this and previous studies are more likely to result from NSAID therapy used to treat the disease than from a disease specific change in bowel flora.

This study has investigated the patterns of membrane 2H4 (CD45RA) and UCHL1 (CD45RO) expression by CD4+ and CD8+ lymphocyte subpopulations in 10 adults and seven children with common variable immunodeficiency (CVI) and X-linked hypogammaglobulinaemia (XLA). Of the 10 adults (CVI, n = 8; XLA, n = 2), only one with a diagnosis of CVI showed normal CD45R expression. For the remaining seven adult CVI patients the abnormal CD45R profiles were primarily associated with CD4+ lymphocytes in three and CD8+ lymphocytes in four. Specific increases in CD45RA- CD45RO+ fractions were found in four CVI patients and in all of these there was a concomitant reduction in circulating CD19+ B-cell numbers. Two of the CVI cases were identical twin sisters and both had the same CD45R abnormality. The two adults with XLA also showed abnormal CD45R expression (increased CD45RA+ CD45RO- components) but of particular note, in view of the fact that these were brothers, one was associated with the CD4+ subpopulation and the other with the CD8+ fraction. Similar analyses for the paediatric group revealed, in distinct contrast to the adult patients, no significant abnormalities of CD45R expression suggesting that these defects may not become apparent until a later age. Further investigations of in vitro lymphocyte proliferation (PHA and PWM) in the eight adults with CVI showed a significant correlation between abnormal responses and disordered CD45R expression. It is proposed that these abnormal patterns of CD45R expression by lymphocyte subpopulations in CVI and XLA may be of fundamental importance with respect to the pathogenesis of these particular immunodeficiencies.

To determine the basis of the tissue cross-reactions shown by some human monoclonal anti-Rh D antibodies, we have investigated the tissue reactivities of 48 further human monoclonal antibodies (mAb) against D and other Rh antigens, and compared them with those of normal and anti-D sera and immunoglobulin preparations, and affinity-purified polyclonal anti-D antibodies. Although we were unable to detect any tissue reactivities associated with the D-binding fraction of polyclonal antisera or prophylactic immunoglobulin, the non-erythroid cell types identified by the tissue-reactive human anti-Rh mAb of both IgM and IgG class were those recognized by antibodies present in both normal and anti-D sera. These results indicate: (a) that the tissue specificities of human anti-Rh mAb are similar to those of natural antibodies, and (b) that there are immunochemical differences between polyclonal and monoclonal anti-D antibodies, at least of IgG class, which may be relevant to the use of the latter in the prevention of haemolytic disease of the new-born by immune prophylaxis.

The differential expression of various membrane CD45R isoforms by normal lymphocyte populations is known to be closely associated with distinct immunoregulatory functions. Abnormal patterns of CD45R expression have been reported in patients with common variable immunodeficiency and HIV infection, and recent evidence has suggested the possibility that one type of variant CD45R expression may be inheritable. By multiple colour flow cytometry, we studied the immunological characteristics of CD4+ helper/inducer and CD8+ suppressor/cytotoxic T cells in a family with variant CD45R expression over three generations. This variant pattern of CD45R expression was shown to affect both CD4+ and CD8+ lymphocyte populations in individual family members and was immunologically characterized by a failure of the normal reciprocal expression of the CD45RA and CD45RO isoforms. Family studies also revealed that this trait had an autosomal dominant mode of inheritance and, in the heterozygous state, appeared not to be associated with major clinical abnormalities. The different isoforms of CD45 show distinct patterns of expression during lymphocyte ontogeny and activation, and these patterns appear to closely reflect function. Despite their diverse immunological roles, our finding that both CD4+ and CD8+ T cells in affected family members showed the same defect suggests a common regulatory mechanism(s) for both these lymphocyte populations. The recognition of this abnormality, particularly in homozygous individuals, will be of great importance in understanding the role of these molecules in immune function and disease.

Sixty-six pre-term infants of less than 30 weeks gestation consecutively admitted to either of two neonatal intensive care units were randomized to receive routine intensive care only or prophylactic intravenous immunoglobulin, 200 mg/kg body weight at 3-weekly intervals in addition to routine intensive care. Eleven babies, 6 in the control group and 5 in the treatment group, were withdrawn from the trial due to early death from extreme prematurity (7 babies), early return to the referring hospital (3 babies), and elective treatment with intravenous immunoglobulin for severe congenital septicaemia (1 baby). Of the 55 who remained in the trial, significantly fewer babies in the treatment group had infection, 8 compared to 17 (p = 0.01). This difference was not significant when blood-culture-proven septicaemia only was considered, 8 compared to 14 (p = 0.09). Twenty-seven (84%) of 32 blood-culture-positive episodes of infection were caused by coagulase-negative staphylococci. Serum IgG was significantly higher with treatment and achieved levels comparable to those of normal full-term babies at the same post-natal age.

In this report the carbohydrate antigens expressed on the three oligosaccharide domains, core, backbone and peripheral, of mucin-type glycoproteins are briefly reviewed in the light of recent observations with monoclonal antibodies. These have revealed that a number of cell-surface antigens which behave as tumour-associated and differentiation antigens of man or mouse are abundantly expressed on the carbohydrate chains of a variety of secreted mucins of human and animal origins and they belong to an antigen system which also includes the major blood group antigens. Examples are given of the use of well-characterized anti-carbohydrate antibodies to derive structural information on (a) mucin-type glycoproteins of human B lymphocyte membranes, (b) the high molecular weight glycoproteins of the normal human gastric and distal-colon mucosae and (c) tumour-derived glycoproteins from these two organs. Major differences between the antigenicities of the normal stomach and distal-colon, and between their tumour-derived glycoproteins, and the important effect of the secretor status in the expression of these antigens are described. These observations have enabled a better understanding of the individual and tissue differences in the expression of tumour-associated antigens. The possibility is raised that these carbohydrate structures (many of which also occur on certain N-linked oligosaccharides and glycolipids) are components of receptor systems for endogenous ligands. More tangible evidence is cited for the role of certain structures in this family of saccharides as receptors for infective agents.

Forty-one patients with acute tuberculous pericarditis were studied retrospectively. Anti-tuberculosis chemotherapy alone was effective in thirty. Five patients died, two from unrelated causes, two due to delayed diagnosis, and one after pericardiectomy. Constrictive pericarditis developed in seven patients, six of whom had successful pericardiectomy. Corticosteroids could not be shown to have reduced the risk of developing constriction. When constriction occurred it did so within the first six months of illness in all cases in contrast to a separate series of 15 patients who presented with constrictive pericarditis. These had had no previous history of tuberculosis, and in 10 cases where pericardiectomy was done, no histological evidence of tuberculosis was found. They were European with an average age of 49 years whereas in the group with acute tuberculous pericarditis 33 were Asian and the average age was 36 years.

Two mouse hybridoma antibodies (LICR-LON-M39 and LICR-LON-M18) against the human-milk-fat globules were found to resemble human autoantibodies of anti-I type in their cold agglutinating property and their preferential reactions with erythrocytes of I- rather than i-type. From inhibition of binding assays with glycoproteins having known A, B, H, Lea, Leb, I, and i activities, and oligosaccharides of the Type 1 and Type 2 lacto-N-glycosyl series, it was established that these antibodies are directed at Type 2 structures, and that the I(Ma) determinant, beta-D-Galp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 6), which is usually found on branched oligosaccharides, is the preferred sequence. The hybridoma antibodies as well as anti-I Ma were shown to react well with the beta-D-Galp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 6)-D-Gal or -D-Man sequence. Studies of the reactions of these antibodies with glycolipids on thin-layer plates showed that the two hybridoma antibodies differ from anti-I Ma in reacting weakly with the unbranched i-type sequence beta-D-Galp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcpNAc-(1 leads to 3)-beta-D-galp-(1 leads to 4) as found on lacto-N-norhexasylceramide. Furthermore, they differ from anti-I Ma but resemble anti-I Woj and Sti, and a hybridoma antibody 1B2 in their failure to react with their determinant in the presence of alpha-D-(1 leads to 3)-linked galactosyl groups. From their lack of reactions with blood-group-A and -H active glycoproteins, and their reactions with neuraminidase-treated erythrocytes, it was deduced that the determinants recognised by the two hybridoma antibodies are also masked in the presence of alpha-L-(1 leads to 2)-linked fucosyl groups and sialic acid.

A monoclonal antibody C14/1/46/10 showing preferential binding to membranes of human colorectal carcinomas over normal colon mucosae was obtained by immunization of mice with extra-nuclear membranes of a human colonic adenoma. Binding and inhibition of binding assays using blood cells or glycoproteins with known blood-group activities indicated that the antibody recognizes a carbohydrate antigen co-existing with the blood-group-H determinant: Fuc alpha 1 leads to 2 Gal. Inhibition assays with structurally defined oligosaccharides showed that the antigenic determinant involves difucosylated Type-2-blood-group chains with the structure: (formula; see text)

The I- and i-antigen activities of chemically synthesized, linear oligosaccharides of the neolacto series containing one, two or three N-acetyllactosamine (Gal beta 1----4GlcNAc) units have been tested by inhibition of binding of five anti-i and eight anti-I monoclonal antibodies to radioiodinated I- and i-active glycoproteins. The inhibitory activities of the milk oligosaccharides lacto-N-neotetraose (Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc) and lacto-N-tetraose (Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc) have also been determined. The results clearly show that: (a) the determinants that best fit the combining sites of anti-i antibodies are at least hexasaccharides of the neolacto series, (b) linear tetra- and hexasaccharides of the neolacto series can strongly inhibit the binding of anti-I antibodies of group 2 which are known to be primarily directed at the repeating Gal beta 1----4GlcNAc beta 1----3 domains of branched neolacto sequences, (c) the beta- but not the alpha-methyl anomer of the glycoside Gal beta 1----4GlcNAc beta 1-O-Me inhibits the binding of anti-I antibodies of group 1 which recognise the branch point sequence Gal beta 1----4GlcNAc beta 1----6-, (d) the reactivity of the beta-methylglycoside is impaired if the sequence is further elongated as in Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-O-Me, and (e) lacto-N-tetraose has no inhibitory activity with any of the anti-i or anti-I antibodies tested.