Rituximab‐induced interleukin‐15 reduction associated with clinical improvement in rheumatoid arthritis

Rituximab therapy alters all aspects of B‐cell participation in the disturbed immune response of rheumatoid arthritis patients. To determine the impact of B‐cell depletion on other immune compartments, we analysed levels of soluble and surface interleukin‐15 (IL‐15) along with the frequency of IL‐15‐related subsets after rituximab treatment. We then studied the correlation of observed changes with clinical activity. Heparinized blood samples from 33 rheumatoid arthritis patients were collected on days 0, 30, 90 and 180 after each of three rituximab cycles. Serum cytokine levels were determined by ELISA. Interleukin‐15 trans‐presentation was analysed by cytometry. Flow cytometry with monoclonal antibodies was performed to analyse circulating cell subsets. Interleukin‐15 was detected in the serum of 25 patients before initiating the treatment. Rituximab then progressively reduced serum IL‐15 (138 ± 21 pg/ml at baseline, 48 ± 18 pg/ml after third cycle, P = 0·03) along with IL‐17 (1197 ± 203 pg/ml at baseline, 623 ± 213 pg/ml after third cycle, P = 0·03) and tended to increase the frequency of circulating regulatory T cells (3·1 ± 1 cells/μl at baseline, 7·7 ± 2 cells/μl after third cycle). Rituximab also significantly decreased IL‐15 trans‐presentation on surface monocytes of patients negative for IL‐15 serum (mean fluorescence intensity: 4·82 ± 1·30 at baseline, 1·42 ± 0·69 after third cycle P = 0·05). Reduction of serum IL‐15 was associated with decrease in CD8⁺ CD45RO⁺/RA⁺ ratio (1·17 ± 0·21 at baseline, 0·36 ± 0·06 at third cycle, P = 0·02). DAS28, erythrocyte sedimentation rate and C‐reactive protein correlated significantly with CD8⁺ CD45RO⁺/RA⁺ ratio (R = 0·323, R = 0·357, R = 0·369 respectively, P < 0·001). Our results suggest that sustained clinical improvement after rituximab treatment is associated with IL‐15/memory T‐cell‐related mechanisms beyond circulating B cells.     

Serum amyloid A induces interleukin‐6 in dermal fibroblasts via Toll‐like receptor 2, interleukin‐1 receptor‐associated kinase 4 and nuclear factor‐κB

Systemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute‐phase protein that is elevated up to 1000‐fold in times of infection or inflammation. This acute‐phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine‐like manner, possibly through toll‐like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin‐6 (IL‐6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL‐6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA‐induced IL‐6 secretion and that this was also mediated through the TLR adaptor protein IL‐1 receptor‐associated kinase 4. The effect is nuclear factor‐κB‐mediated because blockade of nuclear factor‐κB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over‐expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL‐6 signalling in systemic sclerosis via enhanced TLR2 signalling.     

Working memory performance of older adults with insomnia

Older individuals suffering insomnia typically report declines in their cognitive performance beyond what they consider to be normal changes due to the aging process. Recent neuro‐imaging studies have demonstrated frontal lobe hypo‐activation among insomniac populations when compared with healthy, good sleepers. However, research is yet to confirm whether frontal lobe hypo‐activation translates into objective declines when performing tasks hypothesized to draw upon this brain region. This study aimed to investigate whether older insomnia sufferers demonstrate significantly impaired performance on a challenging working memory task when compared with age‐matched good sleepers. Forty‐nine older individuals (mean age = 69.43 years, SD = 4.83) suffering from sleep maintenance insomnia were compared with 49 age‐matched good sleepers. Cognitive performance was assessed using the Double Span Memory Task, a computer‐based working memory task that requires participants to indicate the names and/or spatial locations of increasingly longer sequences of visually presented objects. After controlling for general intelligence, the individuals suffering from insomnia did not perform differently when compared with the good sleepers on either the simpler or more cognitively demanding components of the task. Older individuals with insomnia did not display an observable impairment of working memory in this study relative to good sleepers. Despite the mixed results from previous research, this study adds weight to the absence of objective impairment in insomniacs, at least while performing short‐term demanding cognitive tasks.     

Health Related Quality of Life and Emotional Health in Children with Chronic Granulomatous Disease: A Comparison of Those Managed Conservatively with Those That Have Undergone Haematopoietic Stem Cell Transplant

Purpose

Chronic Granulomatous Disease (CGD) is a rare primary immunodeficiency that predisposes to life-threatening infections and inflammation. Haematopoietic stem cell transplant (HSCT) can cure CGD. Chronic illness reduces quality of life. Children with haematological malignancies report improved quality of life post-HSCT. There are no data for children with CGD. This study evaluated quality of life and emotional well-being in CGD children treated conventionally or transplanted.

Methods

Parents and children completed the Pediatric Quality of Life Inventory v4.0 (PedsQL) and Strengths and Difficulties Questionnaires (SDQ). Mean scores were compared with published UK norms. Comparisons were made for those that had or had not undergone HSCT.

Results

Forty-seven parents completed PedsQL (children aged 3–15). Twenty-one were post-HSCT. Forty-two completed SDQ (children aged 3–15). Nineteen post-HSCT. Median age for non-HSCT group 9 years. Median age for post-HSCT group 10 years. The HSCT group were median 3 years post-HSCT (range 1–9 years). HSCT survival was 90 %—two died without completing questionnaires Parent and self-reported quality of life for non-transplanted children was significantly lower than healthy children. Parents reported increased emotional difficulties compared to published norms. PedsQL and SDQ scores for transplanted children were not significantly different from healthy norms.

Conclusions

This study demonstrates the quality of life is reduced in CGD. Transplanted patients have quality of life comparable to levels reported in healthy children. This data will help inform families and clinicians when deciding about treatment and may have relevance for other immunodeficiencies treated with transplant.     

Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media

Embryos produced by in-vitro fertilization (IVF) may produce less platelet-activating factor (PAF) than is optimal for development. It was previously shown that supplementation of culture media with PAF results in a significant increase in pregnancy rate. Human embryos are often cultured in media supplemented with serum containing the enzyme PAF:acetylhydrolase (PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF. Thus, effective supplementation of media with PAF requires inactivation of this enzyme. In this study we examine the efficacy of the methods of PAF:AH deactivation used for PAF supplementation of IVF culture medium. When the effectiveness of a commonly used acid treatment protocol (pH 3.0 at room temperature for 5 min) was examined, it was found that it was not completely effective for the majority of sera. When synthetic PAF was added to 18 serum samples which had been acid treated, five had 90-100% of the original PAF remaining after 24 h (showing that the acid treatment was effective), eight had from 10-90% of the original PAF remaining after 24 h, and five samples had 0-10%. The extent to which PAF:AH was susceptible to deactivation was not associated with the activity in the serum prior to treatment, the serum oestradiol concentration, or the cause of infertility. The period of acidification and the incubation temperature were assessed to develop a new acid-treatment protocol (20 min acid treatment at 37 degrees C) which was able to deactivate PAF:AH effectively in all sera (53/53) examined. A trial was performed to assess the effect of acid treatment of serum for 5 min at room temperature compared with the new protocol (20 min at 37 degrees C) on IVF outcome, following PAF supplementation of IVF culture medium. Oocyte recovery, fertilization and embryo development rates were equivalent for both groups and approximately equal numbers of embryos were transferred or cryopreserved. Pregnancy rates were not significantly different (14.6 versus 20.0%) for the two treatments, with a trend towards a higher pregnancy rate with the new acid- treatment protocol. The results show that this new procedure for acid treatment of serum in combination with PAF supplementation does not have detrimental effects on embryos and their pregnancy outcome and is therefore suitable for use in IVF.      

Hydrosalpinges adversely affect markers of endometrial receptivity

While in-vitro fertilization (IVF) was initially developed in women with tubal factor infertility, recent clinical studies have suggested that the presence of hydrosalpinges lowers implantation and pregnancy rates. We postulated that these hydrosalpinges cause impaired endometrial receptivity. A total of 103 women with hydrosalpinges were prospectively evaluated, and compared with 55 infertile and 44 fertile controls. All women had endometrial biopsies during the window of implantation, analysed by conventional histological criteria, and also stained for three integrin markers of endometrial receptivity (alpha1beta1, alpha4beta1 and alpha vbeta3). Women with hydrosalpinges (cases) expressed significantly less of the alpha vbeta3 integrin compared with controls. There was no difference in expression of alpha1beta1 or alpha4beta1 among groups. A significantly greater number of cases had out of phase histology and missing alpha vbeta3 (type I defects) and absent integrin expression despite normal histological maturation (type II) defects, compared with controls. Of 20 women with impaired endometrial receptivity who were also biopsied after hydrosalpinx surgery, 70% demonstrated increased alpha vbeta3 expression. Seventy-seven percent of type I and 57% of type II defects were corrected postoperatively. Using markers of endometrial receptivity, this study demonstrates that inflammatory hydrosalpinges have an adverse effect on endometrial receptivity, which in some cases may be overcome by surgical treatment of the hydrosalpinx.      

Somatic mutation processes at a human minisatellite

Germline instability at human minisatellites frequently involves complex inter-allelic transfers of repeat units usually restricted to one end of the repeat array and apparently regulated by flanking DNA. In contrast, nothing is known about the structural basis of somatic instability at minisatellites. An electrophoretic size-enrichment strategy was therefore developed at minisatellite MS32 (D1S8) to enable rare abnormal-length mutants to be detected, validated and quantitated in blood DNA by single molecule PCR. Structural analysis of rare mutant alleles in blood revealed simple deletions/duplications of repeat unit blocks located at random along the tandem repeat array, a mode of mutation completely different from that seen in sperm. Furthermore, allele-specific suppression of sperm instability at MS32 did not affect somatic instability. These data suggest that conversion-based minisatellite mutation in sperm is completely germline-specific and most likely meiotic in origin. Somatic instability appears to occur by a separate pathway involving replication slippage or, more likely, intra-allelic unequal crossing over.      

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB)

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue- specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.