New Frontiers in Autoimmune Diagnostics: A Systematic Review on Saliva Testing.

(1) Background: Immunological laboratory testing is known to be complex, and it is usually performed in tertiary referral centers. Many criticalities affect diagnostic immunological testing, such as limited availability, the need for specifically trained laboratory staff, and potential difficulties in collecting blood samples, especially in the most vulnerable patients, i.e., the elderly and children. For this reason, the identification of a new feasible and reliable methodology for autoantibody detection is urgently needed. (2) Methods: We designed a systematic review to investigate the available literature on the utilization of saliva samples for immunological testing. (3) Results: A total of 170 articles were identified. Eighteen studies met the inclusion criteria, accounting for 1059 patients and 671 controls. The saliva collection method was mostly represented by passive drooling (11/18, 61%), and the most frequently described methodology for antibody detection was ELISA (12/18, 67%). The analysis included 392 patients with rheumatoid arthritis, 161 with systemic lupus erythematosus, 131 with type 1 diabetes mellitus, 116 with primary biliary cholangitis, 100 with pemphigus vulgaris, 50 with bullous pemphigoids, 49 with Sjogren syndrome, 39 with celiac disease, 10 with primary antiphospholipid syndromes, 8 with undifferentiated connective tissue disease, 2 with systemic sclerosis, and 1 with autoimmune thyroiditis. The majority of the reviewed studies involved adequate controls, and saliva testing allowed for a clear distinction of patients (10/12 studies, 83%). More than half of the papers showed a correlation between saliva and serum results (10/18, 55%) for autoantibody detection, with varying rates of correlation, sensitivity, and specificity. Interestingly, many papers showed a correlation between saliva antibody results and clinical manifestations. (4) Conclusions: Saliva testing might represent an appealing alternative to serum-based testing for autoantibody detection, considering the correspondence with serum testing results and the correlation with clinical manifestations. Nonetheless, standardization of sample collection processing, maintenance, and detection methodology has yet to be fully addressed.

Recommendations for the management of mixed cryoglobulinemia syndrome in hepatitis C virus-infected patients.

OBJECTIVE: The objective of this review was to define a core set of recommendations for the treatment of HCV-associated mixed cryoglobulinemia syndrome (MCS) by combining current evidence from clinical trials and expert opinion. METHODS: Expert physicians involved in studying and treating patients with MCS formulated statements after discussing the published data. Their attitudes to treatment approaches (particularly those insufficiently supported by published data) were collected before the consensus conference by means of a questionnaire, and were considered when formulating the statements. RESULTS: An attempt at viral eradication using pegylated interferon plus ribavirin should be considered the first-line therapeutic option in patients with mild-moderate HCV-related MCS. Prolonged treatment (up to 72 weeks) may be considered in the case of virological non-responders showing clinical and laboratory improvements. Rituximab (RTX) should be considered in patients with severe vasculitis and/or skin ulcers, peripheral neuropathy or glomerulonephritis. High-dose pulsed glucocorticoid (GC) therapy is useful in severe conditions and, when necessary, can be considered in combination with RTX; on the contrary, the majority of conference participants discouraged the chronic use of low-medium GC doses. Apheresis remains the elective treatment for severe, life-threatening hyper-viscosity syndrome; its use should be limited to patients who do not respond to (or who are ineligible for) other treatments, and emergency situations. Cyclophosphamide can be considered in combination with apheresis, but the data supporting its use are scarce. Despite the limited available data, colchicine is used by many of the conference participants, particularly in patients with mild-moderate MCS refractory to other therapies. Careful monitoring of the side effects of each drug, and its effects on HCV replication and liver function tests is essential. A low-antigen-content diet can be considered as supportive treatment in all symptomatic MCS patients. Although there are no data from controlled trials, controlling pain should always be attempted by tailoring the treatment to individual patients on the basis of the guidelines used in other vasculitides. CONCLUSION: Although there are few controlled randomised trials of MCS treatment, increasing knowledge of its pathogenesis is opening up new frontiers. The recommendations provided may be useful as provisional guidelines for the management of MCS.

Absence of NF-kappaB activation by a new polystyrene-type adsorbent designed for hemoperfusion.

AIM: The aim of the study was to evaluate biocompatibility of anew polystyrene-type adsorbent (BetaSorb) designed for hemoperfusion, using second-level biomolecular analyses. The device has recently been developed to enhance beta2-microglobulin removal during hemodialysis. Molecular structure and chemical modifications of the surface beads of this cartridge should prevent exposure of dense hydrophobic surface sites to proteins, and avoid the major drawbacks of previous polystyrene-type adsorbent materials. METHODS: Whole blood of healthy donors was incubated in sterile minicolumns packed with BetaSorb Cuprophan, Hemophan, polysulfone and cellulose acetate. In parallel experiments, whole blood was recirculated for 180 min in a sham dialysis circuit equipped with the study sorbent or Hemophan or polysulfone. Biocompatibility was assessed by means of new biomolecular approaches focused on nuclear factor kappaB (NF-kappaB) activation (assessed by electrophoretic mobility shift assay), TNF-alpha and IL-1beta gene expression (evaluated by real-time PCR), TNF-alpha and IL-1beta production (measured by Western blot assay and ELISA), nitric oxide (NO) generation (detected by electron paramagnetic resonance), free oxygen radical production (by chemiluminescence in a biological assay) and the generation of the complement breakdown product C3d. RESULTS: In coincubation experiments, 5-min contact with any dialysis device, but BetaSorb, was enough to induce activation of NF-kappaB. The amount of TNF-alpha precursor form was found to increase after 5 min of exposure to each tested polymer, but no traces of mature forms of TNF-alpha or IL-1beta were detected in in vitro experimental conditions using healthy blood. NO and free oxygen radical generation were significantly lower in blood samples exposed to BetaSorb than in control dialysis devices. C3d levels were found to be increased with Hemophan, unaffected by polysulfone, and remarkably decreased with the BetaSorb device. In the sham hemodialysis experiments, NF-kappaB activation and C3d and NO profiles were similar to direct incubation experiments. Compared to basal levels, quantitation of TNF-alpha and IL-1beta mRNA revealed a 15- and 9-fold increase, respectively, in samples exposed to Hemophan for 180 min. CONCLUSIONS: The new BetaSorb device not only appears to be highly biocompatible, but shares properties that make it probably able to interfere with the activation of the inflammatory state.

Cytokine release pathway in mononuclear cells stimulated in vitro by dialysis membranes.

BACKGROUND: Among the possible variables associated with cytokine activation in hemodialysis, filter membrane has been reported to be a major factor and monocytes adherent to the membrane have been suggested as a possible source for cytokine synthesis. METHODS: In order to exclude variables other than the direct cell-to-membrane interaction, suspensions of peripheral blood monocytes isolated from donors' blood were incubated for 180 min at 37 degrees C in Petri dishes coated with cuprophan (Cu) or polyacrylonitrile (AN69S) or polysulfone (PS). Total RNA was purified, reverse transcribed in cDNA and amplified by polymerase chain reaction (PCR) primed with specific oligomers for determining IL-1beta and TNF-alpha gene expression. For Western blot analysis, cell homogenates and supernatants were electrophoresed and transferred to a polyvinylidene difluoride membrane and the membrane was then incubated with polyclonal antibodies specific for the detection of IL-1beta and TNF-alpha. RESULTS: Semi-quantitation of targets for PCR (using the technique of limiting dilutions and referring to actin and glyceraldehyde-3-phosphate dehydrogenase as noninducible molecule) revealed a comparable increase at 180 min (p < 0.05) in IL-1beta and TNF-alpha mRNA in monocytes incubated with polyacrylonitrile, PS and Cu membranes. In 2 of 10 experiments pro-IL-1beta was detected in monocytes interacting with Cu, PS and AN69S membrane. However, the extent of extracellular release of mature protein was greater for Cu. CONCLUSION: Some dissociation between transcriptional and translational events was detected in experiments using donors' blood in vitro. More specifically, synthetic membranes (both polyacrylonitrile and PS) were found to be as active as Cu in inducing IL-1beta and TNF-alpha mRNA expression, but less effective in promoting the extracellular release of proinflammatory cytokine products.