Enhancing clinical and immunological effects of anti-PD-1 with belapectin, a galectin-3 inhibitor.

BACKGROUND: PD-1/PD-L1 engagement and overexpression of galectin-3 (Gal-3) are critical mechanisms of tumor-induced immune suppression that contribute to immunotherapy resistance. We hypothesized that Gal-3 blockade with belapectin (GR-MD-02) plus anti-PD-1 (pembrolizumab) would enhance tumor response in patients with metastatic melanoma (MM) and head and neck squamous cell carcinoma (HNSCC). METHODS: We performed a phase I dose escalation study of belapectin+pembrolizumab in patients with advanced MM or HNSCC (NCT02575404). Belapectin was administered at 2, 4, or 8 mg/kg IV 60 min before pembrolizumab (200 mg IV every 3 weeks for five cycles). Responding patients continued pembrolizumab monotherapy for up to 17 cycles. Main eligibility requirements were a functional Eastern Cooperative Oncology Group status of 0-2, measurable or assessable disease, and no active autoimmune disease. Prior T-cell checkpoint antibody therapy was permitted. RESULTS: Objective response was observed in 50% of MM (7/14) and and 33% of HNSCC (2/6) patients. Belapectin+pembrolizumab was associated with fewer immune-mediated adverse events than anticipated with pembrolizumab monotherapy. There were no dose-limiting toxicities for belapectin within the dose range investigated. Significantly increased effector memory T-cell activation and reduced monocytic myeloid-derived suppressor cells (M-MDSCs) were observed in responders compared with non-responders. Increased baseline expression of Gal-3+ tumor cells and PD-1+CD8+ T cells in the periphery correlated with response as did higher serum trough levels of pembrolizumab. CONCLUSIONS: Belapectin+pembrolizumab therapy has activity in MM and HNSCC. Increased Gal-3 expression, expansion of effector memory T cells, and decreased M-MDSCs correlated with clinical response. Further investigation is planned.

Omega-3 Fatty Acid Supplementation Improves Endothelial Function in Primary Antiphospholipid Syndrome: A Small-Scale Randomized Double-Blind Placebo-Controlled Trial.

Endothelial cells are thought to play a central role in the pathogenesis of antiphospholipid syndrome (APS). Omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has been shown to improve endothelial function in a number of diseases; thus, it could be of high clinical relevance in APS. The aim of this study was to evaluate the efficacy of n-3 PUFA supplementation on endothelial function (primary outcome) of patients with primary APS (PAPS). A 16-week randomized clinical trial was conducted with 22 adult women with PAPS. Patients were randomly assigned (1:1) to receive placebo (PL, n = 11) or n-3 PUFA (ω-3, n = 11) supplementation. Before (pre) and after (post) 16 weeks of the intervention, patients were assessed for endothelial function (peripheral artery tonometry) (primary outcome). Patients were also assessed for systemic markers of endothelial cell activation, inflammatory markers, dietary intake, international normalized ratio (INR), and adverse effects. At post, ω-3 group presented significant increases in endothelial function estimates reactive hyperemia index (RHI) and logarithmic transformation of RHI (LnRHI) when compared with PL (+13 vs. -12%, p = 0.06, ES = 0.9; and +23 vs. -22%, p = 0.02, ES = 1.0). No changes were observed for e-selectin, vascular adhesion molecule-1, and fibrinogen levels (p > 0.05). In addition, ω-3 group showed decreased circulating levels of interleukin-10 (-4 vs. +45%, p = 0.04, ES = -0.9) and tumor necrosis factor (-13 vs. +0.3%, p = 0.04, ES = -0.95) and a tendency toward a lower intercellular adhesion molecule-1 response (+3 vs. +48%, p = 0.1, ES = -0.7) at post when compared with PL. No changes in dietary intake, INR, or self-reported adverse effects were observed. In conclusion, 16 weeks of n-3 PUFA supplementation improved endothelial function in patients with well-controlled PAPS. These results support a role of n-3 PUFA supplementation as an adjuvant therapy in APS. Registered at http://ClinicalTrials.gov as NCT01956188.

Inflammatory protein response in CDKL5-Rett syndrome: evidence of a subclinical smouldering inflammation.

BACKGROUND: Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT. METHODS: Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated. RESULTS: CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregulation featuring a bulk increase of anti-inflammatory cytokines, predominantly IL-10, could explain the unchanged erythrocyte sedimentation rate and atypical features of inflammation in CDKL5-RTT. Omega-3 PUFAs were able to counterbalance the pro-inflammatory status. CONCLUSION: For the first time, we revealed a subclinical smouldering inflammation pattern in CDKL5-RTT consisting in the coexistence of an atypical APR coupled with a dysregulated cytokine response.

Autoantibodies against homocysteinylated protein in a mouse model of folate deficiency-induced neural tube defects.

BACKGROUND: Periconceptional supplementation with folic acid results in a significant reduction in the incidence of neural tube defects (NTDs). Nonetheless, NTDs remain a leading cause of perinatal morbidity and mortality worldwide, and the mechanism(s) by which folate exerts its protective effects are unknown. Homocysteine is an amino acid that accumulates under conditions of folate-deficiency, and is suggested as a risk factor for NTDs. One proposed mechanism of homocysteine toxicity is its accumulation into proteins in a process termed homocysteinylation. METHODS & RESULTS: Herein, we used a folate-deficient diet in pregnant mice to demonstrate that there is: (i) a significant inverse correlation between maternal serum folate levels and serum homocysteine; (ii) a significant positive correlation between serum homocysteine levels and titers of autoantibodies against homocysteinylated protein; and (iii) a significant increase in congenital malformations and NTDs in mice deficient in serum folate. Furthermore, in mice administered the folate-deplete diet before conception, supplementation with folic acid during the gestational period completely rescued the embryos from congenital defects, and resulted in homocysteinylated protein titers at term that are comparable to that of mice administered a folate-replete diet throughout both the pre- and postconception period. These results demonstrate that a low-folate diet that induces NTDs also increases protein homocysteinylation and the subsequent generation of autoantibodies against homocysteinylated proteins. CONCLUSION: These data support the hypotheses that homocysteinylation results in neo-self antigen formation under conditions of maternal folate deficiency, and that this process is reversible with folic acid supplementation.

Evaluation of the tolerability and immunogenicity of ultraviolet C-irradiated autologous platelets in a dog model.

BACKGROUND: The THERAFLEX ultraviolet (UV) platelets (PLTs) pathogen reduction system for PLT concentrates (PCs) operates using ultraviolet C (UVC) light at a wavelength of 254 nm. UVC treatment can potentially alter proteins, which may affect drug tolerance in humans and influence the immunogenicity of blood products. This preclinical study in beagle dogs was designed to evaluate the safety pharmacology of UVC-irradiated PCs after intravenous administration and to determine whether they are capable of eliciting humoral responses to PLTs and plasma proteins. STUDY DESIGN AND METHODS: Six beagle dogs each were transfused once every other week for 10 weeks with UVC-irradiated or nonirradiated PCs. All PCs were autologous canine single-donor products prepared from whole blood. Safety pharmacology variables were regularly assessed. The impact of UVC irradiation on PLT and plasma proteomes was analyzed by one- and two-dimensional gel electrophoresis. Serum samples were tested for UVC-induced antibodies by Western blot and flow cytometry. RESULTS: Dogs transfused with UVC-irradiated PCs showed no signs of local or systemic intolerance. Few but significant changes in PLT protein integrity were observed after UVC irradiation. Even after repeated administration of UVC-irradiated PCs, no antibodies against UVC-exposed plasma or PLT proteins were detected. CONCLUSIONS: Repeated transfusions of autologous UVC-treated PCs were well tolerated in all dogs studied. UVC irradiation did not cause significant plasma or PLT protein modifications capable of inducing specific antibody responses in the dogs. High-resolution proteomics combined with antibody analysis introduces a comprehensive and sensitive method for screening of protein modifications and antibodies specific for pathogen reduction treatment.

Immune-enhancing effect of Danggwibohyeoltang, an extract from Astragali Radix and Angelicae gigantis Radix, in vitro and in vivo.

Danggwibohyeoltang (DGBHT) is an oriental herbal prescription consisting of two herbs: Astragali Radix and Angelicae gigantis Radix. We examined the immune-enhancing effect of DGBHT in mice using the forced swimming test (FST) and in vitro tests in peritoneal macrophages. After daily oral administration of DGBHT, blood biochemical parameters related to fatigue were measured after the FST. The immobility time in the FST was significantly decreased in the DGBHT-treated group (200 mg/kg) on the 10th and 14th days. DGBHT (100~~200 mg/kg) treatment significantly increased glucose levels, acting as an energy source. Lactic dehydrogenase levels, which are accurate indicators of muscle damage, tended to decline after DGBHT administration (100~200 mg/kg). When DGBHT (200 mg/kg) was orally administered to mice, creatine kinase levels tended to decrease; however, this decrease was not significant. DGBHT did not have any effects on the variation of total protein and blood urea nitrogen levels. Further, we examined how DGBHT regulates cytokine production, nitric oxide (NO) production, and nuclear factor-kappa B (NF-κB) activation in mouse peritoneal macrophages. When DGBHT was used in combination with recombinant interferon-gamma (rIFN-γ), there was a noticeable cooperative induction of NO production and NF-κB activation. Moreover, rIFN-γ plus DGBHT treatment of peritoneal macrophages significantly increased the production of tumor necrosis factor-alpha (TNF-α) and interleukin-12 (IL-12). These results suggest that DGBHT improves immune function through the changes in indicators related to fatigue and the regulatory effects on immunological parameters, such as TNF-α, IL-12, NO production, and NF-κB activation.

Cloning of ovocalyxin-36, a novel chicken eggshell protein related to lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and plunc family proteins.

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20-25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens.

Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy.

Adult stem cells from human bone marrow stroma, referred to as mesenchymal stem cells or marrow stromal cells (hMSCs), are attractive candidates for clinical use. The optimal conditions for hMSC expansion require medium supplemented with fetal calf serum (FCS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FCS proteins. By a sensitive fluorescence-based assay we determined that 7 to 30 mg of FCS proteins are associated with a standard preparation of 100 million hMSCs, a dosage that probably will be needed for clinical therapies. Here we present ex vivo growth conditions for hMSCs that reduce the FCS proteins to less than 100 ng per 100 million hMSCs, approximately a 100,000-fold reduction. The cells maintain their proliferative capacity and sustain their ability for multilineage differentiation. Experiments in rats demonstrate that rat MSCs grown in 20% FCS induce a substantial humoral response after repeated administrations, whereas cells grown under the conditions described in this study reduce the immunogenicity in terms of IgG response over 1000-fold to barely detectable levels. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs and perhaps other cells.

Effects of prebiotics on the immune response to vaccination in the elderly.

BACKGROUND: Prebiotics stimulate the growth of bifidogenic bacteria in the gut. The aim of this work was to assess the effects of a prebiotic mixture on the immune response in healthy elderly people. METHODS: Healthy free-living elderly people (age, > or = 70 years), receiving a nutritional supplement that provided 1.6 MJ, 15 g of protein, and 50% of vitamin daily reference values per day, were randomly assigned to receive a prebiotic mixture (6 g/d of a 70% raftilose and 30% raftiline mixture) or placebo (6 g of maltodextrin powder) for 28 weeks. At week 2 of the study, all subjects were vaccinated with influenza and pneumococcal vaccines. At weeks 0, 2, and 8 of the study, serum total proteins, albumin, immunoglobulins, saliva secretory immunoglobulin A (IgA), and serum titers of influenza A and B and pneumococcal antibodies were measured. At week 8, cultured peripheral monocyte cell secretion of interleukin-4, interferon-gamma, and lymphocyte proliferation, stimulated with phytohemagglutinin and influenza antigen, were measured. RESULTS: Sixty-six subjects were considered eligible for the study, and 43 (20 receiving prebiotics and 23 receiving placebo) were considered for final analyses on a per protocol basis. No changes in serum proteins, albumin, immunoglobulins, and secretory IgA were observed. Antibodies against influenza B and pneumococcus increased significantly from weeks 0 to 8, with no significant differences between groups. Antibodies against influenza A did not increase. No effects of prebiotics on interleukin-4 and interferon-gamma secretion by cultured monocytes were observed. CONCLUSIONS: No immunological effects of prebiotics were observed in this study.

The influence of a residual group in low-molecular-weight allergoids of Artemisia vulgaris pollen on their allergenicity, IgE- and IgG-binding properties.

BACKGROUND: Reaction of epsilon-amino groups of lysine with potassium cyanate, maleic, or succinic anhydride leads to allergoids of low molecular weight. No study has been performed to compare their properties and investigate the influence of a residual group on allergenicity and human IgE- and IgG-binding of these derivatives. METHODS: Allergoids of a pollen extract of Artemisia vulgaris were obtained by means of potassium cyanate, and succinic and maleic anhydride. Biochemical properties were investigated by determination of amino groups, enzyme activity, isoelectric focusing IEF and SDS-PAGE. IgE- and IgG-binding was determined using immunoblots and ELISA inhibition. Allergenicity was investigated by skin prick tests (SPT) on a group of 52 patients, of which 6 were control subjects, 30 were patients with no previous immunotherapy (IT), and 16 were patients undergoing immunotherapy. RESULTS: The same degree of amino-group modification (more than 85%), residual enzyme activity (less then 15%), IEF, and SDS-PAGE pattern were noted. In the immunoblots of IgE-binding, there was more pronounced reduction in the succinyl and maleyl derivatives than in the carbamyl one. IgG-binding was less affected by carbamylation than by acid anhydride modification. The SPT showed that the succinylated derivative had the most reduced allergenicity (98% showed a reduced wheal diameter when tested with the succinyl derivative, 87% with the maleyl allergoid, and 83% with the carbamyl allergoid). The most significant difference among allergoids could be seen in the group of patients with high skin reactivity (83% of patients showed no reaction to the succinyl derivative when compared to the value of 28% for the carbamyl derivative or 22% for the maleyl derivative). CONCLUSIONS: According to our results, all three modification procedures yielded allergoids with a similar extent of modification. No single biochemical parameter investigated in the study could predict the degree of reduced allergenicity in vivo. The most reduced allergenicity was seen in the succinyl derivative while the preservation of IgG binding epitopes was of the highest degree for the carbamyl derivative.

Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy.

The first approved clinical gene therapy trial for adenosine deaminase (ADA) deficiency employed autologous T cells grown in fetal calf serum (FCS)-supplemented medium and transduced with a retroviral vector (LASN) also produced in the presence of FCS. Ten years after their enrollment, both patients have circulating T cells containing vector DNA. However, whereas approximately 20% of the circulating T cells from patient 1 are still vector positive, less than 1% of patient 2's T cells have detectable vector. This difference appears to be not only a function of the original transduction efficiency and cell expansion capability in vitro, but also of the immune response that patient 2 developed to FCS components during the course of her treatment. In this study, serum samples from each patient were tested for antibodies to FCS by enzyme-linked immunosorbent assay and anti-FCS responses were demonstrated in both patients. Analysis of immunoglobulin classes revealed comparable levels of IgA and IgM anti-FCS titers. Patient 2, however, had significantly higher IgG responses to FCS than did patient 1. Investigation of the development of anti-FCS responses by IgG subclasses indicated that there was a different pattern in the development of IgG immunity to FCS between the two patients. In addition, significant antibody response to bovine lipoprotein was detected in patient 2, but not in patient 1 or in control samples. These findings suggest that the unique immune response mounted by patient 2 may have influenced the outcome of the gene transfer treatments in this patient.

[The influence of a three-year preseasonal specific immunotherapy on selected parameters of allergic inflammation in pollinosis patients]. / Wplyw trzyletniej przedsezonowej immunoterapii swoistej na wybrane parametry zapalenia alergicznego u chorych na pylkowice.

Specific immunotherapy (SIT) can in some cases influence the course of allergic inflammation (eosinophilia and ECP concentration in peripheral tissue). This study was set up to evaluate the efficacy of three-year pre-seasonal SIT with grass pollen allergoid. We measured NALf eosinophilia and ECP concentration both in NALf and blood serum after subsequent SITs. Twenty seven patients aged 26.7 +/- 7.4 (range 18-45) entered this study. They were randomly assigned to treatment with either Pollinex or Allergovit. We observed a progressive fall in NALf eosinophilia in subsequent years: 24.1 +/- 2.4%; 20.2 +/- 4.6%; 9.8 +/- 1.9% vs. 30.4 +/- 3.0% before treatment (p < 0.05, p < 0.05 and p < 0.001, respectively). Also ECP concentration fell after second and third SIT to 15.6 +/- 1.5 ng/ml i 12.96 +/- 1.75 ng/ml vs 23.3 +/- 3.7 ng/ml before SIT (p < 0.05). A significant drop in serum ECP concentration was recorded only after the third SIT season--2.5 +/- 1.23 micrograms/ml vs 5.8 +/- 1.3 micrograms/ml before treatment, p < 0.01. NALf eosinophilia correlated positively with NALf ECP concentration--R2 = 0.92, p < 0.05. Hence, SIT ameliorates allergic inflammation decreasing significantly activity of eosinophils in nasal mucosa measured as NALf eosinophilia and ECP concentration. This effect seems to be time-depended.

Prime solutions for cardiopulmonary bypass in neonates: antioxidant capacity of prime based on albumin or fresh frozen plasma.

OBJECTIVE: Oxidative damage and inflammation are believed to play an important role in postoperative complications after cardiopulmonary bypass. During bypass, a prime solution with a high antioxidant capacity may reduce the oxidative damage and inflammation. We investigated total antioxidant capacity and individual scavengers during the preparation of 2 different prime solutions. METHODS: The prime solutions were prepared with either pasteurized human albumin or fresh frozen plasma. The total antioxidant capacity was measured with the total radical antioxidant parameter assay and with the ferric-reducing ability of plasma assay. The individual scavengers vitamin C, sulfhydryl groups, uric acid, and total protein were measured before, during, and after the prime preparation. Malondialdehyde was measured as a parameter for lipid peroxidation. RESULTS: Neither prime solution showed a total radical antioxidant parameter value. The ferric-reducing ability of plasma value of prime solutions was lower than that of undiluted human albumin or fresh frozen plasma. Addition of mannitol did not increase the ferric-reducing ability of plasma value. Vitamin C was only found in the fresh frozen plasma prime. Both prime solutions contained sulfhydryl groups and uric acid in low concentrations. During ultrafiltration, low-molecular-weight antioxidants were lost into the ultrafiltrate. CONCLUSIONS: We showed that prime solutions based on either albumin or fresh frozen plasma had very low antioxidant capacity and that ultrafiltration of the prime solution further lowers this capacity. A prime solution with a low antioxidant capacity may increase oxidative stress in neonates undergoing cardiopulmonary bypass.

Mucosal output of eotaxin in allergic rhinitis and its attenuation by topical glucocorticosteroid treatment.

BACKGROUND: Eotaxin is a chemokine that attracts and activates eosinophils. The present study examines the occurrence of eotaxin in nasal mucosal surface liquids in patients with seasonal allergic rhinitis without allergen exposure and during repeat allergen challenge with and without topical glucocorticosteroid treatment. The number of subepithelial eosinophils and mucosal outputs of bulk plasma (alpha2-macroglobulin) and eosinophil cationic protein (ECP) are also examined. METHODS: Twelve patients underwent daily allergen challenges for 6 days. Separately, 14 patients, who were receiving budesonide and placebo in a parallel group design, also underwent allergen challenge for 6 days. Nasal biopsies were obtained before and 24 h after the allergen challenge series, and lavages were carried out before and 15 min after selected allergen challenges. RESULTS: At baseline nasal lavage fluid levels of eotaxin correlated to levels of alpha2-macroglobulin and ECP. After the first allergen challenge there was a correlation between nasal lavage fluid levels of eotaxin and ECP. Repeat allergen exposure increased the mucosal output of eotaxin (P <0.05) and ECP (P <0.01) as well as eosinophil numbers (P <0.01), but no correlation was found between increased eosinophil numbers and eotaxin. Budesonide reduced eotaxin levels during repeat allergen challenge (P <0.05). CONCLUSIONS: Repeat allergen exposure in allergic rhinitis is associated with increased mucosal output of eotaxin. Topical budesonide attenuates this effect, suggesting the possibility that inhibitory effects on mucosal eotaxin may contribute to anti-eosinophilic actions of topical glucocorticosteroids.

The endotoxin-binding bactericidal/permeability-increasing protein (BPI): a target antigen of autoantibodies.

The bactericidal/permeability-increasing protein (BPI) is an endotoxin-binding neutrophil leukocyte-granule protein with antibacterial and anti-endotoxin properties. A recombinant form of BPI (rBPI21) has been developed and is being tested as a therapeutic agent to treat gram-negative bacterial infections and exposure to gram-negative bacterial endotoxin. BPI is also a target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA). BPI-ANCA are present in cystic fibrosis, inflammatory bowel disease, vasculitis, and primary sclerosing cholangitis; presence of BPI-ANCA appears associated with a higher inflammatory disease activity and greater organ damage. BPI-ANCA as well as ANCA directed at other neutrophil-granule proteins may exacerbate inflammation by nonspecific effects of extracellular and cell-associated immune complexes. BPI-ANCA may further worsen inflammation by reducing the ability of BPI to promote clearance of gram-negative bacteria and bacterial-associated endotoxin.

Evidence for local eosinophil differentiation within allergic nasal mucosa: inhibition with soluble IL-5 receptor.

Eosinophil differentiation occurs within the bone marrow in response to eosinopoietic cytokines, particularly IL-5. Recently, however, eosinophil precursors (CD34/IL-5Ralpha+ cells) and IL-5 mRNA+ cells have been identified within the lungs of asthmatics, indicating that a population of eosinophils may differentiate in situ. In this report, we examined the presence of eosinophil precursors within allergic nasal mucosa and examined whether they undergo local differentiation following ex vivo stimulation. We cultured human nasal mucosa obtained from individuals with seasonal allergic rhinitis with either specific allergen, recombinant human IL-5 (rhIL-5), or allergen + soluble IL-5Ralpha (sIL-5Ralpha), shown to antagonize IL-5 function. Simultaneous immunocytochemistry and in situ hybridization demonstrated that there were fewer cells coexpressing CD34 immunoreactivity and IL-5Ralpha mRNA following culture with allergen or rhIL-5, compared with medium alone. Immunostaining revealed that the number of major basic protein (MBP) immunoreactive cells (eosinophils) was higher within tissue stimulated with allergen or rhIL-5, compared with unstimulated tissue. In situ hybridization detected an increase in IL-5 mRNA+ cells in sections from tissue cultured with allergen, compared with medium alone. These effects were not observed in tissue cultured with a combination of allergen and sIL-5Ralpha. Colocalization analysis indicated this expression to be mainly, but not exclusively, T cell (44%) and eosinophil (10%) derived. Our findings suggest that a subset of eosinophils may differentiate locally within allergic nasal mucosa, in what appears to be a highly IL-5-dependent fashion, and imply that this process might be regulated in vivo by endogenous production of sIL-5Ralpha.

Postoperative blood salvage and reinfusion in spinal surgery: blood quality, effectiveness and impact on patient blood parameters.

Although reinfusion of salvaged shed blood has become popular in major orthopaedic procedures, this blood saving technique is still controversial. In an effort to assess the functional and metabolic status of shed blood erythrocytes and the impact of postoperative shed blood reinfusion on allogenic blood requirements and patient's blood parameters, analyses of perioperative blood samples were performed in 28 consecutive orthopaedic patients undergoing spinal fusion, in which postoperative shed blood was collected and reinfused with the ConstaVac CBC II device. In comparison with a previous series of 31 patients, this procedure reduced allogenic blood requirements by almost 30% (P < 0.05), without any increase in postoperative complications. Postoperative shed blood presented lower haematological values and higher plasma-free haemoglobin (PFHB) levels than preoperative blood, without any disturbance in morphology, median corpuscular fragility (MCF) or erythrocyte adenosine triphosphate (ATP) and diphosphoglycerate (DPG) content. Serum concentrations of enzymes--glutamate-oxalacetate aminotransferase (GOT), glutamate-piruvate aminotransferase (GPT), creatine kinase (CK), lactate dehydrogenase (LDH)--and inflammatory cytokines (IL-1beta, IL-6) were elevated in shed blood. After reinfusion, there was no alteration in coagulation parameters or cytokine levels. Serum levels of some enzymes increased at the end of surgery and remained elevated at postoperative day 2 (CK) or 7 (GOT, LDH), with a higher increase if postoperative autotransfusion was used as a blood saving method. Therefore, caution should be taken when these serum enzyme levels are used for diagnosis. In conclusion, salvaged shed blood in orthopaedic procedures of the spine seems to be an excellent source of red cells which are not significantly damaged, keeping a normal functional and metabolic status, and reduces allogenic blood requirements without significant side effects.

Sensitivity to the house dust mite and airway hyperresponsiveness in a young adult population.

BACKGROUND: The pathogenic mechanisms of airway hyperresponsiveness (AHR) in asthma are unknown and only a few studies have examined the importance of sensitivity to antigens in AHR in young adults. OBJECTIVE: We investigated the correlation between AHR and sensitivity to specific antigens, atopy, history of childhood asthma and spirometry in a young adult population. METHODS: Based on the results of interviews with 447 students at our university, 308 non-smoker students were classified into six groups. Group 1 comprised subjects with intermittent mild bronchial asthma; group 2, subjects with history of childhood asthma; group 3, subjects with atopic disease, and a RAST score for Dermatophagoides farinae (Def) of > or = 2; group 4, normal subjects with a RAST score for Def of > or = 2; group 5, subjects with cedar pollinosis; and group 6, normal subjects. We measured AHR to methacholine (MCh), spirometry, immunoglobulin E-radioimmunosorbent test (IgE-RIST), IgE-radioallergosorbent test to six common antigens, eosinophil cationic protein (ECP), and eosinophil count in peripheral blood in each subject. RESULTS: Airway hyperresponsiveness to MCh did not correlate with IgE-RIST, eosinophil count, or ECP. The highest AHR to MCh was present in groups 1 and 2 and lowest in groups 5 and 6. Multiple regression analysis showed that sensitivity to Def was the only factor that significantly influenced AHR to MCh. Airway hyperresponsiveness to MCh of groups with a RAST score for Def of 0/1 was lower than groups with a RAST score of 2 to 6. Airway hyperresponsiveness to MCh did not correlate with the degree of positivity to Def antigen among positive sensitized groups (RAST score 2 to 6). CONCLUSIONS: Sensitivity to mite antigen may be important in the pathogenesis of AHR and Def is a major contributing antigen in young adults in Japan. Once asthma occurs, AHR remains positive for a long time even after the disappearance of asthma-related symptoms.

Asthma, bronchial hyperreactivity and mediator release in children with birch pollinosis. ECP and EPX levels are not related to bronchial hyperreactivity.

BACKGROUND: Symptoms of allergic asthma are triggered by allergen exposure inducing allergic inflammation and hyperreactivity of the bronchi. OBJECTIVES: To investigate the possible relationship between clinical symptoms and signs of asthma, i.e. bronchial variability as measured by peak expiatory flow rate (PEFR), bronchial hyperreactivity (BHR) and mediators of allergic inflammation. METHODS: Twenty-eight children with pollinosis, but no obvious history of asthma, were studied at three occasions, i.e. before, during and after (autumn) the birch pollen season. Twelve children sensitive to birch pollen were considered as the case group. Sixteen children, who were only clinically sensitive to grass pollen, served as controls. Subjective symptoms of asthma were recorded by visual analogue scale, BHR was estimated by methacholine bronchial provocation tests, bronchial variability PEFR and circulating mediators of inflammation, i.e. eosinophil cationic protein, eosinophil protein X, myeloperoxidase and tryptase in serum. RESULTS: Bronchial hyperreactivity and by PEFR was more pronounced after than during the season (P < 0.01), whereas eosinophil mediators and the peak expiratory flow rate increased during the season (P < 0.05). Except for between PEFR variability and BHR in the autumn (r = 0.45; P = 0.014), no correlations were found. However, in the autumn, the majority of children were still hyperreactive in the bronchi and showed PEFR variability but the levels of eosinophil mediators in serum had returned to normal levels. CONCLUSION: Signs and symptoms of asthma did not correlate with serum levels of mediators of allergic inflammation. Bronchial hyperreactivity and PEFR variability persisted after the pollen season when signs of bronchial inflammation had disappeared. We hypothesize that eosinophil mediators and other markers of allergic inflammation disappear after the late-phase reaction, whereas BHR persists. This would explain the lack of correlation between the levels of eosinophil mediators in serum and symptoms of asthma and BHR.