NLRP3 (rs10754558) gene polymorphism and tumor necrosis factor alpha as predictors for disease activity and response to methotrexate and adalimumab in psoriasis.

BACKGROUND: Psoriasis has a global prevalence of 1-3%, with variations observed across different ethnic groups and geographical areas. Disease susceptibility and response to anti-tumor necrosis factor-α (TNFα) drugs suggest different genetic regulatory mechanisms which may include NLR family pyrin domain containing 3 (NLRP3) polymorphism. Evaluation of the NLRP3 gene polymorphism, the serum level of CRP and TNFα in psoriasis patients and assessment of the NLRP3 (rs10754558) gene polymorphism, CRP and TNFα with disease severity and their role as biomarkers for response to Methotrexate and Adalimumab in psoriasis. The study had a total of 75 patients diagnosed with psoriasis vulgaris, who were compared to a control group of 75 healthy individuals. RESULTS: There was a highly significant difference in NLRP3 genotypes and alleles distribution between psoriasis patients and controls (P = 0.002,0.004). The heterozygote genotype GC (OR = 3.67,95%CI:1.75-7.68, P = 0.0006), was linked with increased risk of psoriasis. Additionally, The GC genotype was significantly associated with nonresponse to psoriasis therapy (OR = 11.7,95%CI:3.24-42.28, P = 0.0002). Regarding serum CRP and TNFα levels, there was a highly statistically significant difference between psoriasis patients and controls (P < 0.0001), and there was also a highly statistically significant difference between responders and non-responders in psoriasis patients regarding PASI 50 (P < 0.0001). CONCLUSIONS: The NLRP3 (rs10754558) genotypes GC was associated with the severe form of psoriasis and with nonresponse to psoriasis medication. Therefore, NLRP3 (rs10754558) gene polymorphism is an important prognostic biomarker in psoriasis patients. The serum TNFα can be used as a predictor for response to therapy in psoriasis patients. More research for evaluation of role of the NLRP3 gene polymorphism in the genetic risks and treatment outcomes associated with psoriasis is still required.

Immunogenetic Relationship of HLA-G 14 bp Insertion/Deletion Polymorphism and Toll-Like Receptor 9 with Systemic Lupus Erythematosus in Egyptian Patients: A Case-Control Study.

INTRODUCTION: The level of expression of the immunoregulatory human leukocyte antigen-G (HLA-G) has been suggested to play a role in the immunopathogenesis of systemic lupus erythematosus (SLE). A 14 bp insertion/deletion (ins/del) polymorphism in the 3'untranslated region of HLA-G gene may influence the level of expression. The role of Toll-like receptor 9 (TLR9) in the pathogenesis of SLE has been highlighted. Data among Egyptian patients are quite limited. PURPOSE: To detect the association of HLA-G 14 bp ins/del gene polymorphism with the susceptibility to SLE and to correlate TLR9 serum level with disease activity among Egyptian patients. PATIENTS AND METHODS: A case-control study that included 102 SLE female patients and 102 healthy matched volunteers as controls was carried out. Disease activity in patients was determined using the modified Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). HLA-G 14 bp ins/del genotype was detected by polymerase chain reaction (PCR). TLR9 serum level was estimated using enzyme-linked immunosorbent assay (ELISA) technique. RESULTS: The ins/ins genotype was significantly increased among SLE patients compared to healthy subjects (58.8% vs 9.8%; odds ratio [OR] = 11.79, P < 0.001). The 14 bp ins allele was significantly more frequent in SLE patients than in healthy subjects (65.7% vs 27.9%, respectively) and significantly associated with an increased risk of SLE (OR 4.94, P < 0.001). The mean TLR9 serum level showed a significant increase in SLE patients compared to healthy subjects (397.04±137.86 vs 195.22±45.14 ng/L, p < 0.001) and was significantly associated with disease activity as well as to patients' HLA-G genotypes (p < 0.001). CONCLUSION: Among Egyptian population, HLA-G 14 bp ins/ins homozygous genotype and ins allele may constitute a potential risk for SLE susceptibility, while TLR9 serum level is significantly associated with disease activity.

Seronegative autoimmune hepatitis in children : A real diagnostic challenge.

BACKGROUND AND AIM: Classical autoimmune hepatitis (AIH) is characterized by the presence of conventional autoantibodies (anti-smooth muscle, antinuclear and anti-liver-kidney-microsomal antibodies). The absence of such autoantibodies in some patients does not preclude AIH diagnosis or the need for its treatment. This group of patients was termed seronegative AIH. Whether non-conventional autoantibodies can identify this group of patients is still elusive. We aimed to study the prevalence of seronegativity of conventional autoantibodies and the occurrence of non-conventional autoantibodies in children with AIH. METHODS: In this study, 55 children with AIH were investigated for non-conventional autoantibodies (anti-neutrophil cytoplasmic antibodies, antibodies to soluble liver antigen, anti-tissue transglutaminase and antiplatelet antibodies). All the patients received immunosuppressive therapy and were assessed for treatment response. RESULTS: Of the patients 44 had classical AIH (type 1, 70.09%, type 2, 9.09%) and 20% were seronegative. The four studied non-conventional autoantibodies occurred in four patients, one for each. All non-conventional autoantibodies were exclusively associated with type 1 AIH. The clinical profile, ultrasonographic findings, liver biochemistry and histopathological findings were comparable in the classical and seronegative AIH. The majority of patients with classical (72.7%) and seronegative (54.5%) AIH were treatment responders. CONCLUSION: Seronegative AIH represents a substantial percentage of pediatric patients diagnosed with AIH. They were even negative for non-conventional autoantibodies. Furthermore, apart from autoantibodies, seronegative AIH is almost indistinguishable from the classical AIH and the majority of patients were treatment responders. This favorable response to immunosuppression deserves sustainable efforts for considering such a diagnosis and start therapy to halt disease progression is worthwhile.

Role of mannose binding lectin in response to candida antigen immunotherapy of warts.

BACKGROUND: Warts is the commonest cutaneous manifestation of human papillomavirus (HPV) infection. Intralesional Candida antigen immunotherapy is used for wart treatment. AIM: To identify the role of mannose binding lectin (MBL) in susceptibility to HPV infection and to explore the relationship between MBL and response to intralesional Candida antigen immunotherapy of wart. PATIENTS AND METHODS: A case-control study was enrolled with 96 participants; 48 wart cases and 48 healthy controls. MBL serum level assay baseline and after six settings of intralesional candida antigen injection was done by ELISA technique. MBL2 gene exon 1 codon 54 polymorphism was detected by using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). RESULTS: A statistically significant difference in MBL serum level between wart cases and controls was found. An association between MBL2 exon1 codon 54 polymorphism and susceptibility to HPV infection and development of warts was proved. Carriage of genotype AB was more frequent wart cases (95.8%) than in controls (20.8%). No statistical significance association could be found between the therapeutic response to Candida antigen immunotherapy in wart cases and MBL as regards its serum level and genotypes. CONCLUSIONS: MBL play an important role in host defense against HPV infection.

The Role of the Level of Interleukin-33 in the Therapeutic Outcomes of Immunotherapy in Patients with Allergic Rhinitis

Abstract Introduction Allergic rhinitis (AR) affects up to 40% of the population and results in nasal itching, congestion, sneezing, and clear rhinorrhea. Objectives This study aimed to evaluate the changes in the clinical symptoms and in the level of serum interleukin (IL)-33 before and after pollen immunotherapy (IT) in patients with AR. Methods The total symptomscore and the levels of total immunoglobulin E (IgE) and IL-33 were determined in the serum of 10 non-allergic healthy controls and 45 patients with AR who were equally divided into 3 groups: GI (patients did not receive IT), GII (patients had received IT for 6 months) and GIII (patients had received IT for 2 years). Results There was a significantly higher concentration of IgE and IL-33 in the serum of patients with AR than in that of non-allergic patients. Furthermore, serum level of IL-33 decreased significantly after pollen IT. But, there was no significant reduction in the serum level of IL-33 between GII and GIII patients. Conclusion Our results show a clinical improvement associated with a decrease in serum level of IL-33 after pollen IT.

The Role of the Level of Interleukin-33 in the Therapeutic Outcomes of Immunotherapy in Patients with Allergic Rhinitis.

Introduction Allergic rhinitis (AR) affects up to 40% of the population and results in nasal itching, congestion, sneezing, and clear rhinorrhea. Objectives This study aimed to evaluate the changes in the clinical symptoms and in the level of serum interleukin (IL)-33 before and after pollen immunotherapy (IT) in patients with AR. Methods The total symptom score and the levels of total immunoglobulin E (IgE) and IL-33 were determined in the serum of 10 non-allergic healthy controls and 45 patients with AR who were equally divided into 3 groups: GI (patients did not receive IT), GII (patients had received IT for 6 months) and GIII (patients had received IT for 2 years). Results There was a significantly higher concentration of IgE and IL-33 in the serum of patients with AR than in that of non-allergic patients. Furthermore, serum level of IL-33 decreased significantly after pollen IT. But, there was no significant reduction in the serum level of IL-33 between GII and GIII patients. Conclusion Our results show a clinical improvement associated with a decrease in serum level of IL-33 after pollen IT.

APRIL Level as a Marker of Disease Activity in Treated Rheumatoid Arthritis Patients: Association with Disease Activity and Anti-CCP Antibody.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with joint inflammation and autoantibody production. Cytokines play an important role in the pathogenesis of RA. Among the cytokines that regulate B cell homeostasis is the "A Proliferation-Inducing Ligand" (APRIL). To determine the differences in APRIL in response to treatment in anti- cyclic citrllinated peptides (anti-CCP) positive versus anti-CCP negative patients with established RA. Concentrations of APRIL in sera of 10 anti-CCP positive RA patients, 18 anti-CCP negative RA patients, and 12 healthy controls were measured by enzyme-linked immunosorbent assay (ELISA) at treatment initiation and after 6 months of treatment with methotrexate and hydroxychloroquine. Correlations between serum anti-CCP, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), 28-joint Disease Activity Score (DAS28), and serum level of APRIL were analyzed. Serum APRIL levels were increased in rheumatoid arthritis patients in comparison with healthy volunteers. APRIL correlated positively with disease activity; swollen joint count, visual analog score and simplified disease activity index (all P < 0.05). In addition, APRIL was significantly higher in patients with positive anti-CCP. After treatment, APRIL levels significantly decreased in the anti-CCP positive RA patients than in anti-CCP negative RA patients. In conclusions, serum APRIL may be a good predictor marker for joint injury and therapeutic response in patients with RA.

Amniotic membrane graft to conjunctival flap in treatment of non-viral resistant infectious keratitis: a randomised clinical study.

PURPOSE: To evaluate and compare the results of bipedicle conjunctival flap (CF) and cryopreserved amniotic membrane graft (AMG) in the treatment of non-viral infectious keratitis resistant to medical treatment. METHODS: This prospective randomised interventional study included 40 eyes of 40 patients with resistant non-viral infectious keratitis. Twenty eyes received CF and 20 eyes received AMG. In the CF group, there were 12 eyes with fungal keratitis, 7 eyes with bacterial keratitis and 1 eye with Acanthamoeba keratitis. In the AMG group there were 13 eyes with fungal keratitis, 5 eyes with bacterial keratitis and 2 eyes with Acanthamoeba keratitis. In the CF group, three ulcers had descemetocele and four ulcers were perforated. In the AMG group, four ulcers had descemetocele and two ulcers were perforated. In CF, 360° peritomy was done and a bipedicle CF from the upper conjunctiva was dissected from Tenon's capsule, mobilised to cover the cornea and sutured to episclera. In AMG, one or two layers of AM were trimmed to fit the ulcer and sutured to the cornea. The follow-up period was 6 months. RESULTS: Successful results were observed in 18/20 eyes (90%) in each group. Postoperatively, no significant differences between the two groups were found regarding success rate (p=1.0), epithelialisation time (p=0.75) or visual acuity improvement (p=0.84). CONCLUSIONS: CF and AMG are effective in treatment-resistant infectious keratitis. They could restore ocular surface integrity and provide metabolic and mechanical support for corneal healing. For large corneal perforation, it may be better to use another procedure such as penetrating keratoplasty to restore ocular integrity.

Differential expression of Toll-like receptors 7 & 8 mRNA in monocytes of patients with chronic hepatitis C infection: correlation with interferon and ribavirin treatment.

Toll-like receptors (TLRs) have been identified as key regulators of innate and adaptive immune responses in viral infection. The contribution of the host immune response to sustained virologic response is not clear in patients with chronic hepatitis C virus (HCV) infection. This study aimed to investigate the expression of TLRs 7 & 8 mRNA in monocytes of patients with chronic (HCV) infection showing different responses to interferon and ribavirin treatment. The study group was comprised of 21 chronic HCV infected patients. mRNA levels of TLRs 7 & 8 in monocytes were evaluated using real-time PCR before a 48-week treatment with pegylated interferon (PEG-IFN) alpha-2b and ribavirin. In addition, we studied TNF production in monocytes using three-color immunofluorescence and flow cytometry after TLRs 7 & 8 ligand (R848) stimulation. According to the virological outcome of the treatment, the infected patients were classified into nonresponders (NR) and sustained virological responders (SVR). The study determined that mRNA levels of TLRs 7 & 8 were significantly high in SVR compared to NR. Furthermore, after stimulation with R848 the median fluorescence intensity for TNF protein was significantly high in SVR compared to NR. Our data show that a differential mRNA expression of TLRs 7 & 8 is associated with different responses to IFN-based antiviral therapy in patients with chronic HCV. These findings suggest that the TLRs-expression profiles of monocytes from patients with chronic HCV may be useful biomarkers for IFN therapy.

Increased serum APRIL differentially correlates with distinct cytokine profiles and disease activity in systemic lupus erythematosus patients.

Cytokines play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Among the cytokines that regulate B cell homeostasis is a proliferation-inducing ligand (APRIL). This study aimed to determine whether serum levels of APRIL are raised in patients with SLE and correlate with disease activity or proinflammatory cytokines production, or both. Serum APRIL, interleukin-17 (IL-17), IL-4 and interferon gamma (IFN-γ) levels were measured in forty patients with SLE and 30 healthy controls. Disease activity was assessed by SLE disease activity index (SLEDAI), and results were correlated with serum APRIL levels. Serum APRIL levels were significantly higher in patients with SLE than in healthy controls. Positive correlation was found between serum APRIL levels and total SLEDAI score and anti-dsDNA antibody titers. Moreover, serum APRIL levels was significantly higher in patients with arthritis, mucocutaneous manifestations and proteinuria. APRIL is increased in patients with active SLE accompanying the increase of IL-17 and IFN-γ. Significant positive correlations between serum levels of APRIL and IL-17 and IFN-γ and a negative correlation between serum levels of APRIL and IL-4 were found. The results suggest that APRIL may be an important marker of disease activity in patients with SLE. We provide the analyses of APRIL levels in patients with SLE, suggesting new tools for the diagnosis, prognosis and possible therapeutic management of SLE.

R848, a Toll-like receptors 7 and 8 agonist, a potential therapy for allergic rhinitis patients.

BACKGROUND/PURPOSE(S): There is a growing interest in the targeting of Toll-like receptors (TLRs) for the treatment of allergic diseases. TLRs7/8 ligands are future candidates of therapeutic value in allergic rhinitis (AR). This study focus on TLRs7/8 ligand; resiquimod (R848) as an adjuvant to immunotherapy (IT) in AR patient. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from atopic donors and non atopic donors. PBMCs were cultured in the absence and presence of date palm pollen allergen (Phoenix dactylifera; Pho d) and/or R848. Interleukin-4 (IL-4), IL-10, IL-13 and interferon gamma (IFN-γ) were measured in the culture supernatants. RESULTS: R848 was able to significantly increase the anti-inflammatory response in atopic donors more than non atopic donors. Nevertheless, the combination of both; R848 and Pho d provides inferior stimulus as compared to R848 alone in both atopic and non atopic donors. CONCLUSION: Invitro treatment of PBMCs with R484 hijacks the pro inflammatory immune process triggered by TLRs7/8 to mediate anti-inflammatory response. This may provide a conception about the activity and efficacy of TLRs7/8 ligands in AR and open the gate for them to be applied in clinically in humans.

Immunomodulatory effect of R848 on cytokine production associated with Schistosoma mansoni infection.

This work aimed to determine the effect of resiquimod (R848), a low molecular weight imidazoquinolinamine compound with properties as immune response modifiers, on cytokine production in patients infected with Schistosoma mansoni with various degrees of hepatic fibrosis. Schistosoma-infected patients were classified into four groups according to the degree of hepatic fibrosis as examined by ultrasonography group 0 (no fibrosis), group I (incipient fibrosis), group II (moderate fibrosis), and group III (severe fibrosis). Levels of interferon-γ (IFN-γ), interleukin 4 (IL-4), IL-10, and IL-13 were measured by ELISA in the supernatants of cultured peripheral blood mononuclear cells (PBMC) in response to schistosoma egg soluble antigen (SEA) in the presence and absence of R848. R848 induced a significantly higher level of IL-10 in supernatants of SEA-stimulated PBMC of patients in all groups of hepatic fibrosis except group III. Moreover, we significantly found higher levels of IFN-γ in group 0 and group I and significantly lower level of IL-4 in group 0 after R848 stimulation. However, R848 had no effect on IL-13. Taken together, these results suggest that R848 might be used as an immunopotentiating agent in view of novel therapy or vaccine for S. mansoni infection.

Immunological markers in allergic rhinitis patients treated with date palm immunotherapy.

OBJECTIVE: Allergic rhinitis (AR) is regulated by the local production and release of several cytokines. Allergen specific immunotherapy (IT) has been widely used for many years as a specific treatment of allergic diseases. This study aimed to investigate the changes in clinical and immunological markers before and after Phoenix dactylifera IT in AR patients. MATERIALS AND METHODS: Total symptom score and levels of total immunoglobulin E (IgE), albumin, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-4, and IL-13 were measured in the serum and nasal samples of thirty non-atopic healthy controls and thirty patients with allergic rhinitis before and after 15 months of IT. RESULTS: We found significantly higher concentrations of serum TNF-α and nasal TNF-α and IL-13, and significantly lower concentration of nasal IL-10, in allergic patients than in non-allergic. Moreover, serum and nasal IL-10 increased significantly after IT. However, there was neither a significant reduction in total IgE nor a significant increase in IFN-γ at the end of IT. CONCLUSIONS: Our data show a clinical improvement associated with a decline in some inflammation parameters after IT. Moreover, date palm IT induced a significant increase in serum and nasal IL-10 levels.

Laboratory diagnosis of human brucellosis in Egypt and persistence of the pathogen following treatment.

INTRODUCTION: Brucellosis is a major public health problem in Egypt. The Brucella IgM/IgG lateral flow assay was developed as a point-of-care test for the diagnosis of human brucellosis. The aim of this study was to assess the diagnostic value of the lateral flow assay for use in Egypt. METHODOLOGY: Fifty samples of patients who presented with clinical suspicion of brucellosis over a one-year period were collected. All samples were subjected to the Brucella IgM/IgG lateral flow assay, serum agglutination test (SAT), rose bengal RB Test (RB), 2- mercapteoethanol (2-ME), culture and PCR. SAT, 2- ME, culture and PCR were retested after the end of the treatment. RESULTS: Culture and SAT confirmed the diagnosis of brucellosis in twenty patients. While 90% of the samples were positive by SAT, only 30% and 85% were positive by culture and PCR respectively. The sensitivity of the lateral flow assay calculated for the Brucella IgM/IgG was 95% and specificity was 97%. CONCLUSION: These data show that the lateral flow assay is more suitable for diagnosis of brucellosis in Egypt than culture and SAT. Application of the PCR on serum samples collected during follow-up revealed that the DNA of the pathogen was yet not completely cleared almost 60 days after the start of treatment with doxycycline and ciprofloxacin.

Standardized single-platform assay for human monocyte subpopulations: Lower CD14+CD16++ monocytes in females.

We present a novel single-platform assay for determination of the absolute number of human blood monocyte subpopulations, i.e., the CD14(++)CD16(-) and the CD14(+)CD16(++) monocytes. A four-color combination of antibodies to CD14, CD16, CD45, and HLA-DR reduces the spill-over of natural killer cells and of granulocytes into the CD14(+)CD16(++) monocyte gate. For these CD14(+)CD16(++) monocytes, the intra-assay coefficient of variation (CV) was 4.1% and the inter-assay CV was 8.5%. Looking at a cohort of 40 donors aged 18-60 years, we found no age dependence. There was however an effect of gender in that females had lower CD14(+)CD16(++) monocytes (45.4 +/- 13.5 cells/microl) compared with males (59.1 +/- 20.3 cells/microl) (P < 0.02). Using this novel approach, we can confirm that exercise will lead to more than three-fold increase of the CD14(+)CD16(++) monocytes. Also, we show that therapy with low doses of glucocorticoids will deplete these cells. This robust single-platform assay may be a useful tool for monitoring the absolute number of monocyte subpopulations in health and disease.

Hyposensitization in the treatment of resistant cases of vernal keratoconjunctivitis.

PURPOSE: The study evaluated the treatment of resistant cases of vernal keratoconjunctivitis by hyposensitization resulting from intradermal skin reactions of different allergens. METHODS: This prospective study was carried out in the Department of Ophthalmology and the Department of Microbiology and Immunology, Zagazig University, and included 36 patients with bilateral vernal keratoconjunctivitis that was resistant to topical corticosteroids and antihistaminic drugs. Patients were subjected to intradermal skin reactions to different allergens after stoppage of the medication. Subcutaneous injections of different allergens were administered in addition to topical vasoconstrictor and antihistaminic eye drops for different durations of 12, 18, and 24 months. RESULTS: The study revealed that most of the patients were sensitive to pollens (65%), house dust (55%), and tobacco smoke (40%). Among 36 total patients who received different doses of allergens, 10 of 20 patients showed marked improvement following a 24-month treatment period, with a 50% success rate. Another 3 of 9 patients showed marked improvement following an 18-month treatment period, with a 33.3% success rate. CONCLUSION: The use of intradermal skin reactions to determine the sensitivity for different stimulating allergens in resistant cases of vernal keratoconjunctivitis was conclusive. Treatment by hyposensitization using prepared vaccines had an acceptable success rate especially in patients treated for 24 months. Future studies will be needed to determine how effective this treatment is with other allergens and for longer durations of hyposensitization. Additional immunologic studies will be essential for developing a strategy of management of resistance in such cases of resistant vernal keratoconjunctivitis.