Microcirculation in the foot is augmented by neuromuscular stimulation via the common peroneal nerve in different lower limb postures: a potential treatment for leg ulcers.

OBJECTIVES: To examine the effects of the geko™ device (a portable electical nerve stimulator) on microcirculatory flow on the dorsum of the foot, and whether this is influenced by lower limb postures and application of a plaster cast. STUDY DESIGN: Cross-sectional, healthy cohort, open label, physiological response study. METHODS: In 10 healthy volunteers, aged 19 to 24 years, Laser Doppler Fluxmetry measurements were made on the dorsum of the foot in four postures: standing (weight bearing and non-weight bearing) and supine lying (with the lower limb horizontal and then elevated). Measurements of flux were made both at rest and during stimulation with the geko™ device applied over the common peroneal nerve, at 1Hz for 5 minutes in each posture. Repeat measurement were made after the application of a below knee plaster cast. Measures of flux were compared to basal levels assumed to be in supine with limb horizontal, with no cast and an inactive Geko device. RESULTS: The geko™ device was effective in increasing microcirculation on the dorsum of the foot in all four postures (Mean difference =141%, 95% CI 70%-212%, p=0.001). This effect was more pronounced than that of using a plaster alone (Mean increase in Flux of 73%, 95% CI 22%-125%, p=0.01) or variances due to the hydrostatic effects of different postures (Mean difference 17-27.6%, p>0.05). There was a 2 to 3 fold increase in flux when stimulation was delivered in combination with the plaster cast. CONCLUSIONS: Stimulation using the geko™ device augments microcirculation in the foot. The response is greater in lying and non-weight bearing than weight bearing standing but the most striking effect is when stimulation is combined with a plaster cast. The geko™ offers a potential means of promoting conditions favourable for wound healing, where treatment using compression may be contraindicated, such as arterial/mixed aetiology ulcers.

Microcirculation in the foot is augmented by neuromuscular stimulation via the common peroneal nerve in different lower limb postures: a potential treatment for leg ulcers.

AIM: Aim of the study was to examine the effects of the geko™ device (a portable electical nerve stimulator) on microcirculatory flow on the dorsum of the foot, and whether this is influenced by lower limb postures and application of a plaster cast. METHODS: This was a cross-sectional, healthy cohort, open label, physiological response study. In 10 healthy volunteers, aged 19 to 24 years, laser Doppler fluxmetry measurements were made on the dorsum of the foot in four postures: standing (weight bearing and non-weight bearing) and supine lying (with the lower limb horizontal and then elevated). Measurements of flux were made both at rest and during stimulation with the geko™ device applied over the common peroneal nerve, at 1 Hz for 5 minutes in each posture. Repeat measurement were made after the application of a below knee plaster cast. Measures of flux were compared to basal levels assumed to be in supine with limb horizontal, with no cast and an inactive geko™ device. RESULTS: The geko™ device was effective in increasing microcirculation on the dorsum of the foot in all four postures (mean difference =141%, 95% CI 70%-212%, P=0.001). This effect was more pronounced than that of using a plaster alone (Mean increase in flux of 73%, 95% CI 22%-125%, P=0.01) or variances due to the hydrostatic effects of different postures (mean difference 17-27.6%, P>0.05). There was a 2 to 3 fold increase in flux when stimulation was delivered in combination with the plaster cast. CONCLUSION: Stimulation using the geko™ device augments microcirculation in the foot. The response is greater in lying and non-weight bearing than weight bearing standing but the most striking effect is when stimulation is combined with a plaster cast. The geko™ offers a potential means of promoting conditions favourable for wound healing, where treatment using compression may be contraindicated, such as arterial/mixed aetiology ulcers.

Neuromuscular electrostimulation viathe common peroneal nerve promotes lower limb blood flow in a below-kneecast: A potential for thromboprophylaxis.

OBJECTIVES: We aimed to examine the characteristics of deep venous flow in the leg in a cast and the effects of a wearable neuromuscular stimulator (geko; FirstKind Ltd) and also to explore the participants' tolerance of the stimulator. METHODS: This is an open-label physiological study on ten healthy volunteers. Duplex ultrasonography of the superficial femoral vein measured normal flow and cross-sectional area in the standing and supine positions (with the lower limb initially horizontal and then elevated). Flow measurements were repeated during activation of the geko stimulator placed over the peroneal nerve. The process was repeated after the application of a below-knee cast. Participants evaluated discomfort using a questionnaire (verbal rating score) and a scoring index (visual analogue scale). RESULTS: The geko device was effective in significantly increasing venous blood flow in the lower limb both with a plaster cast (mean difference 11.5 cm/sec(-1); p = 0.001 to 0.13) and without a plaster cast (mean difference 7.7 cm/sec(-1); p = 0.001 to 0.75). Posture also had a significant effect on peak venous blood flow when the cast was on and the geko inactive (p = 0.003 to 0.69), although these differences were less pronounced than the effect of the geko (mean difference 3.1 cm/sec(-1) (-6.5 to 10)). The geko device was well tolerated, with participants generally reporting only mild discomfort using the device. CONCLUSION: The geko device increases venous blood flow in the lower limb, offering a potential mechanical thromboprolylaxis for patients in a cast. Cite this article: Bone Joint Res 2013;2:179-85.

[Interleukin-1 cytokines, inflammasomes, NOD-signalosomes and autoinflammation]. / Interleukin-1-Zytokine, Inflammasome, NOD-Signalosome und Autoinflammation.

The understanding of the genetic and immunological basis of human periodic fever syndromes, in particular cryopyrin-associated periodic syndromes (CAPS), has led to important new insights into the pathogenesis of monogenic and complex interleukin-1beta-associated autoinflammatory diseases. Currently the focus of attention is on the nucleotide-binding oligomerization domain (NOD)-like receptors (NLR), which take part in the regulation of the synthesis and maturation of cytokines in the IL-1 families, NOD-signalosomes and inflammasomes.

Pregnancy induces numerical and functional changes of CD4+CD25 high regulatory T cells in patients with rheumatoid arthritis.

OBJECTIVE: In a prospective study we investigated whether numerical and functional changes of CD4+CD25(high) regulatory T cells (Treg) were associated with changes of disease activity observed during pregnancy and post partum in patients with rheumatoid arthritis (RA). METHODS: The frequency of CD4+CD25(high) T cells was determined by flow cytometry in 12 patients with RA and 14 healthy women during and after pregnancy. Fluorescence-activated cell sorting (FACS) was used to sort CD4+CD25(high) T cells and CD4+CD25- T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies alone or in co-culture to investigate proliferation and cytokine secretion. RESULTS: Frequencies of CD4+CD25(high) Treg were significantly higher in the third trimester compared to 8 weeks post partum in patients and controls. Numbers of CD4+CD25(high) Treg inversely correlated with disease activity in the third trimester and post partum. In co-culture experiments significantly higher amounts of IL10 and lowered levels of tumour necrosis factor (TNF)alpha and interferon (IFN)gamma were found in supernatants of the third trimester compared to postpartum samples. These findings were independent from health or disease in pregnancy, however postpartum TNFalpha and IFN gamma levels were higher in patients with disease flares. CONCLUSION: The amelioration of disease activity in the third trimester corresponded to the increased number of Treg that induced a pronounced anti-inflammatory cytokine milieu. The pregnancy related quantitative and qualitative changes of Treg suggest a beneficial effect of Treg on disease activity.

Detection of latent tuberculosis in immunosuppressed patients with autoimmune diseases: performance of a Mycobacterium tuberculosis antigen-specific interferon gamma assay.

OBJECTIVE: To analyse the performance of a new M. tuberculosis-specific interferon gamma (IFNgamma) assay in patients with chronic inflammatory diseases who receive immunosuppressive drugs, including tumour necrosis factor alpha (TNFalpha) inhibitors. METHODS: Cellular immune responses to the M. tuberculosis-specific antigens ESAT-6, CFP-10, TB7.7 were prospectively studied in 142 consecutive patients treated for inflammatory rheumatic conditions. Results were compared with tuberculin skin tests (TSTs). Association of both tests with risk factors for latent M. tuberculosis infection (LTBI) and BCG vaccination were determined and the influence of TNFalpha inhibitors, corticosteroids, and disease modifying antirheumatic drugs (DMARDs) on antigen-specific and mitogen-induced IFNgamma secretion was analysed. RESULTS: 126/142 (89%) patients received immunosuppressive therapy. The IFNgamma assay was more closely associated with the presence of risk factors (odds ratio (OR) = 23.8 (95% CI 5.14 to 110) vs OR = 2.77 (1.22 to 6.27), respectively; p = 0.009), but less associated with BCG vaccination than the TST (OR = 0.47 (95% CI 0.15 to 1.47) vs OR = 2.44 (0.74 to (8.01), respectively; p = 0.025). Agreement between the IFNgamma assay and TST results was low (kappa = 0.17; 95% CI 0.02 to 0.32). The odds for a positive IFNgamma assay strongly increased with increasing prognostic relevance of LTBI risk factors. Neither corticosteroids nor conventional DMARDs significantly affected IFNgamma responses, but the odds for a positive IFNgamma assay were decreased in patients treated with TNFalpha inhibitors (OR = 0.21 (95% CI 0.07 to 0.63), respectively; p = 0.006). CONCLUSIONS: These results demonstrate that the performance of the M. tuberculosis antigen-specific IFNgamma ELISA is better than the classic TST for detection of LTBI in patients receiving immunosuppressive therapy for treatment of systemic autoimmune disorders.

[Adult-onset primary immunodeficiencies]. / Adult-manifestierende primäre Immundefizienzen.

Different individuals with the same kind of primary immunodeficiency may start having symptoms from early childhood on, or alternatively much later in adult life, or never. The differences in phenotype can only partly be deduced from genotype-analysis or--in case of female patients with X-linked diseases--from age-related skewing of lyonisation. The role of compensatory immune mechanisms is less clear. The microbial spectrum of infections is usually the same for both adult and infantile forms of a special primary immunodeficiency syndrome. Yet, many of the adult forms are associated with non-infectious complications, such as granuloma formation, autoimmunity or tumors. Besides standard antibiotic treatment and IgG replacement therapy, there are now different cytokine- or enzyme-replacement regimens available for some of the primary immunodeficiencies. However, exact diagnostic classification of the immunodeficiency should be obtained before such treatment modalities are used. Adult primary immunodeficiency syndromes therefore represent a challenge to both clinicians and molecular biologists.

BPI-ANCA in transporter associated with antigen presentation (TAP) deficiency: possible role in susceptibility to Gram-negative bacterial infections.

Although HLA class I expression is diminished in patients with defects in the transporter associated with antigen presentation (TAP), recurrent Gram-negative bacterial lung infections are found from childhood onwards. As MHC class II-mediated responses are normal, other mechanisms that contribute to susceptibility to infections are presumed. The bactericidal/permeability-increasing protein (BPI) is a potent neutrophil antibiotic that neutralizes endotoxin efficiently. As antineutrophil cytoplasmic autoantibodies (ANCA) against BPI were found in the majority of cystic fibrosis patients and correlate with disease severity we examined the prevalence of BPI-ANCA and their contribution to susceptibility to bacterial infections in six TAP-deficient patients. Although only two patients showed ANCA in indirect immunofluorescence, BPI-ANCA occurred in five of six patients in ELISA. Purified IgG from BPI-ANCA-positive sera (five of six) inhibited the antimicrobial function of BPI in vitro. Epitope mapping revealed binding sites not only on the C-terminal but also on the antibiotic N-terminal portion of BPI, indicating that short linear BPI peptide fragments may be long-lived enough to become immunogens. In conclusion, BPI-ANCA are associated strongly with TAP deficiency. Inhibition of the antimicrobial BPI function by BPI-ANCA demonstrates a possible mechanism of how autoantibodies may contribute to increased susceptibility for pulmonary Gram-negative bacterial infections by diminished bacterial clearance.

Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography.

CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipid specific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone- and foldase-assisted refolding of denatured CD1d molecules and beta(2)-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-alpha-galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples. Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.

Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40.

The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.

Association of a syndrome resembling Wegener's granulomatosis with low surface expression of HLA class-I molecules.

BACKGROUND: Granulomatous syndromes, such as Wegener's granulomatosis, are defined according to complex criteria, but the underlying cause is rarely identified. We present evidence for a new aetiology for chronic granulomatous lesions associated with a recessive genetic defect, which is linked to the human leucocyte antigen (HLA) locus. METHODS: Five adults with necrotising granulomatous lesions in the upper respiratory tract and skin, associated with recurrent bacterial respiratory infections and skin vasculitis, were identified. A diagnosis of Wegener's granulomatosis was considered in all of them, but abandoned because of an incompatible disease course and resistance to immunosuppressive treatments. Peripheral-blood samples were taken and analysed by immunohistochemistry and fluorescent-activated-cell-sorter analysis. Since all five patients were homozygous for the HLA locus, we looked for genetic defects located within the HLA-locus with PCR and restriction fragment length polymorphism. FINDINGS: A severe decrease in cell-surface expression of HLA class-I molecule was seen in all patients. Defective expression of the transporter associated with antigen presentation (TAP) genes was responsible for the HLA class-I down-regulation, and in two patients we identified a mutation in the TAP2 gene responsible for the defective expression of the TAP complex. We showed the presence of autoreactive natural killer (NK) cells and gammadelta T lymphocytes in the peripheral blood cells of two patients. Correction of the genetic defect in vitro restored normal expression of HLA class-I molecules and prevented self-reactivity in the patients' cells. Histology of granulomatous lesions showed the presence of a large proportion of activated NK cells. INTERPRETATION: Our findings define the cause and pathogenesis of a new syndrome that affects patients with a defective surface expression of HLA class-I molecules. The syndrome resembles Wegener's granulomatosis both clinically and histologically. Patients have chronic necrotising granulomatous lesions in the upper respiratory tract and skin, recurrent infections of the respiratory tract, and skin vasculitis. A predominant NK population within the granulomatous lesions suggests that the pathophysiology of the skin lesions may relate to the inability of HLA class-I molecules to turn off NK cell responses. Accurate genetic analysis of a defined syndrome can provide a better understanding of the cause and pathogenesis of a disease.

A case of primary immunodeficiency due to a defect of the major histocompatibility gene complex class I processing and presentation pathway.

INTRODUCTION: We report a case of primary immunodeficiency due to a defect of the TAP transporter, an heterodimeric complex which controls the expression of HLA class I molecule by delivering peptides from the cytosol into the lumen of the endoplasmic reticulum. Since childhood, the 36 year old female suffered from recurrent sinusitis/bronchitis. She later developed bronchiectasis and destructive nasal epitheloid granulomata in conjunction with a generalized vasculitic syndrome that did not improve upon immunosuppression and antibiotics. METHODS: The class I monomorphic W6/32 was used for cell surface staining and immunoprecipitation of MHC class I molecules. Peptide transport assay was carried out in semi-permeabilized cells with iodinated peptides. Antigen presentation experiments were performed using chromium 51 labelled patient B cell line and EBV specific CTL. TAP1 and TAP2 specific antibodies were used for Western blotting and immunoprecipitation of the TAP complex. RESULTS AND CONCLUSIONS: A severe reduction of MHC class I molecules at the cell surface of the B-cell lines was observed, whereas MHC class II expression was not altered. Isoelectric focusing of metabolically labelled MHC class I molecules revealed that class I heavy chains remain unsialylated, consistent with a block of TAP dependent peptide translocation. These conclusions were confirmed by further experiments showing that peptide translocation was completely abolished. We also demonstrated that presentation of viral antigens through endogenous class I molecules was severely impaired. Immunoprecipitation and Western blotting of TAP1/2 complex showed that TAP2 was not detectable. Further, experiments are in progress to identify the site of the mutation.