Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Transitional cell carcinoma of the ovary: A rare case and review of literature

Goblet cell carcinoid of the large intestine is a rare neoplasm, usually located in ascending colon and rectum. A 60-year-old male patient underwent surgery after the diagnosis of acute abdomen. Exploratory laparotomy revealed perforation with a diameter of 1 cm at the site of the previously performed gastroenterostomy and dilatation of the right colic flexure, secondary to a solid obstructive mass located in the mid-portion of transverse colon. Histopathological investigation of the biopsies, taken from the gastroenterostomy site and the tumor, revealed mixed carcinoid-adenocarcinoma with carcinoid component, predominantly composed of goblet cells. Three cycles of FOLFOX-4 protocol was administered. Following respiratory distress secondary to pulmonary metastasis, the patient's condition deteriorated and subsequently died in the fourth postoperative month. Our aim with this paper is to point out that more cases should be reported for more effective diagnosis, histopathological study, clinical investigation, treatment and prognosis of this specific neoplasm.     

Prospective cohort studies on risk factors for cardiovascular events in systemic lupus erythematosus: a major challenge

Cardiovascular disease (CVD) has been identified as a major contributor to morbidity and mortality in patients with systemic lupus erythematosus (SLE). The etiology of premature CVD in SLE is supposed to have many factors, including traditional coronary artery disease (CAD) risk factors, antiphospholipid antibodies, and metabolic and inflammatory factors. Despite the overwhelming interest in CVD in SLE research, prospective studies evaluating risk factors for hard endpoints (that is, cardiovascular events) are relatively scarce. The article by Gustafsson and colleagues suggests that prothrombotic factors play an important role in SLE-related CVD and that the influence of traditional CAD risk factors might be limited.     

Polygalacturonase inhibiting protein in plant cell walls

Polygalacturonase inhibiting protein (PGIP) is localized in plant cell walls and plays an important role both in pectic substance metabolism and in prevention of the penetration of phytopathogenic microorganisms. Apparently, PGIP is responsible for the specificity of cell--cell interactions during pollination or inoculation by fungi nonpathogenic for the particular plant. PGIPs from different plants share a basic common structure. They are rather thermostable glycoproteins enriched with leucine and contain about 20% carbohydrates; the molecular weight varies between 37-54 kD. The synthesis of PGIP is encoded by one gene, and its expression is stimulated by injury and fungal infection. The resistance of plant tissues to infection frequently correlates with PGIP expression and with inhibiting action on fungal PG. Thus, PGIP is believed to be useful for gene engineering to obtain transgenic plants resistant to fungal infection or retaining commercial value during storage.     

Rhythmic activities of isolated and clustered pacemaker neurons and photoreceptors of Aplysia retina in culture

Each eye of Aplysia contains a circadian clock that produces a robust rhythm of optic nerve impulse activity. To isolate the pacemaker neurons and photoreceptors of the eye and determine their participation in the circadian clock and its generation of rhythmic autoactivity, the retina was dissociated and its cells were placed in primary cell culture. The isolated neurons and photoreceptors survived and vigorously extended neurites tipped with growth cones. Many of the photoreceptors previously described from histological sections of the intact retina were identified in culture, including the large R-type photoreceptor, which gave robust photoresponses, and the smaller tufted, whorled, and flared photoreceptors. The pacemaker neurons responsible for the rhythmic impulse activity generated by the eye were identified by their distinctive monopolar morphology and recordings were made of their activity. Isolated pacemaker neurons produced spontaneous action potentials in darkness, and pacemaker neurons attached to fragments of retina or in an isolated cluster interacted to produce robust spontaneous activity. This study establishes that isolated retinal pacemaker neurons retain their innate autoactivity and ability to produce action potentials in culture and that clusters of coupled pacemaker neurons are capable of generating robust autoactivity comparable to pacemaker neuron rhythmic activity recorded in the intact retina, which was previously shown to correspond to 1:1 with the optic nerve compound action potential activity. © 1996 John Wiley & Sons, Inc.     

Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis

Antibodies to citrullinated proteins (anti-cyclic-citrullinated peptide [anti-CCP] antibodies) are highly specific for rheumatoid arthritis (RA) and precede the onset of disease symptoms, indicating a pathogenetic role for these antibodies in RA. We recently showed that distinct genetic risk factors are associated with either anti-CCP-positive disease or anti-CCP-negative disease. These data are important as they indicate that distinct pathogenic mechanisms are underlying anti-CCP-positive disease or anti-CCP-negative disease. Likewise, these observations raise the question of whether anti-CCP-positive RA and anti-CCP-negative RA are clinically different disease entities. We therefore investigated whether RA patients with anti-CCP antibodies have a different clinical presentation and disease course compared with patients without these autoantibodies. In a cohort of 454 incident patients with RA, 228 patients were anti-CCP-positive and 226 patients were anti-CCP-negative. The early symptoms, tender and swollen joint count, and C-reactive protein level at inclusion, as well as the swollen joint count and radiological destruction during 4 years of follow-up, were compared for the two groups. There were no differences in morning stiffness, type, location and distribution of early symptoms, patients' rated disease activity and C-reactive protein at inclusion between RA patients with and without anti-CCP antibodies. The mean tender and swollen joint count for the different joints at inclusion was similar. At follow-up, patients with anti-CCP antibodies had more swollen joints and more severe radiological destruction. Nevertheless, the distribution of affected joints, for swelling, bone erosions and joint space narrowing, was similar. In conclusion, the phenotype of RA patients with or without anti-CCP antibodies is similar with respect to clinical presentation but differs with respect to disease course.     

Design of reference populations for genomic selection in crossbreeding programs

Background

In crossbreeding programs, genomic selection offers the opportunity to make efficient use of information on crossbred (CB) individuals in the selection of purebred (PB) candidates. In such programs, reference populations often contain genotyped PB animals, although the breeding objective is usually more focused on CB performance. The question is what would be the benefit of including a larger proportion of CB individuals in the reference population.

Methods

In a deterministic simulation study, we evaluated the benefit of including various proportions of CB animals in a reference population for genomic selection of PB animals in a crossbreeding program. We used a pig breeding scheme with selection for a moderately heritable trait and a size of 6000 for the reference population.

Results

Applying genomic selection to improve the performance of CB individuals, with a genetic correlation between PB and CB performance (r_PC) of 0.7, selection accuracy of PB candidates increased from 0.49 to 0.52 if the reference population consisted of PB individuals, it increased to 0.55 if the reference population consisted of the same number of CB individuals, and to 0.60 if the size of the CB reference population was twice that of the reference population for each PB line. The advantage of using CB rather than PB individuals increased linearly with the proportion of CB individuals in the reference population. This advantage disappeared quickly if r_PC was higher or if the breeding objective put some emphasis on PB performance. The benefit of adding CB individuals to an existing PB reference population was limited for high r_PC.

Conclusions

Using CB rather than PB individuals in a reference population for genomic selection can provide substantial advantages, but only when correlations between PB and CB performances are not high and PB performance is not part of the breeding objective.     

Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis

MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.     

Anti-citrullinated fibronectin antibodies in rheumatoid arthritis are associated with human leukocyte antigen-DRB1 shared epitope alleles

Introduction

Fibronectin is one of the most abundant proteins present in the inflamed joint. Here, we characterized the citrullination of fibronectin in the joints of rheumatoid arthritis (RA) patients and studied the prevalence, epitope specificity and human leukocyte antigen (HLA) association of autoantibodies against citrullinated fibronectin in RA.

Methods

Citrullinated residues in fibronectin isolated from RA patient synovial fluid were identified by mass spectrometry. The corresponding citrullinated and non-citrullinated peptides were synthesized and used to analyze the presence of autoantibodies to these peptides in RA sera and sera from other diseases and healthy controls by ELISA. The data were compared with risk factors like shared epitope HLA alleles and smoking, and with clinical features.

Results

Five citrullinated residues were identified in fibronectin from RA synovial fluid. RA sera reacted in a citrulline-dependent manner with two out of four citrullinated fibronectin peptides, one of which contains two adjacent citrulline residues, in contrast to non-RA sera, which were not reactive. The most frequently recognized peptide (FN-Cit_1035,1036, LTVGLTXXGQPRQY, in which × represents citrulline) was primarily targeted by anti-CCP (cyclic citrullinated peptide) 2-positive RA patients. Anti-FN-Cit_1035,1036 autoantibodies were detected in 50% of established anti-CCP2-positive RA patients and in 45% of such patients from a early arthritis clinic. These antibodies appeared to be predominantly of the immunoglobulin G (IgG) isotype and to be associated with HLA shared epitope alleles (odds ratio = 2.11).

Conclusions

Fibronectin in the inflamed synovia of RA patients can be citrullinated at least at five positions. Together with the flanking amino acids, three of these citrullinated residues comprise two epitopes recognized by RA autoantibodies. Anti-citrullinated fibronectin peptide antibodies are associated with HLA shared epitope alleles.