Humoral and cellular responses to cow milk proteins in patients with milk-induced IgE-mediated and non-IgE-mediated disorders

BACKGROUND: Cow milk allergy (CMA) is one of the most common food allergies in childhood. Patients with CMA present with a wide range of immunoglobulin (Ig)E- and non-IgE-mediated clinical syndromes. Limited information is known about the specific humoral and cellular responses to cow milk proteins in these various forms of CMA. OBJECTIVE: The aim of the study was to determine IgE, IgA, IgG1 and IgG4 antibody levels and lymphocyte proliferative responses to the major cow milk allergens in patients with IgE- and non-IgE-mediated CMA. METHODS: One hundred and forty cow milk allergic patients, 6 months to 22 years of age, were included in the study. One hundred and thirteen patients had IgE-mediated CMA, 11 had milk protein-induced enterocolitis syndrome and 16 had allergic eosinophilic gastroenteritis. Twenty-one patients without food allergy, 8 months to 18 years of age, served as controls. Serum IgE, IgA, IgG1 and IgG4 antibodies to alpha-, beta-, and kappa-casein, alpha-lactalbumin and beta-lactoglobulin were measured using enzyme-linked immunosorbent assays. For a subset of these patients, we performed lymphocyte proliferation assays to the various milk allergens. RESULTS: Patients with IgE-mediated CMA had higher specific IgE concentrations to casein compared with whey proteins (P < 0.001). In this group of patients, there was a positive correlation between IgE levels and levels of the other isotypes for all four milk proteins (P < 0.001). In general, the caseins were the more allergenic and antigenic proteins in all groups of patients. Patients with enterocolitis syndrome produced less milk protein-specific IgG4 (P < 0.05) and had a trend for higher IgA antibody levels when compared to the control group. Lymphocyte proliferative responses in all groups with CMA were significantly higher than controls (P < 0.05), although this response was similar in patients with IgE- and non-IgE-mediated CMA. CONCLUSION: There is a distinct pattern of humoral antibody response in the different forms of CMA. Patients with IgE-mediated CMA have an elevated polyisotypic response to cow milk protein. The relative lack of specific IgG4 production in patients with enterocolitis syndrome may be involved in the pathogenesis of the disease. In general, caseins appear to be the predominant allergen in patients with CMA.     

CD28 expression is increased in venom allergic patients but is not modified by specific immunotherapy

Background Recognition of antigen bound to the major histocompatibility complex by the T cell receptor is insufficient to lead to T cell proliferation and effector function, which require co-stimulatory signals, such as those resulting from the interaction of CD28 expressed on T lymphocytes and CD80/CD86 expressed on APCs. Lack of interaction between these accessory molecules during antigen stimulation leads to a state of antigen-specific lymphocyte unresponsiveness. Previous studies have shown that rush venom immunotherapy decreases venom-specific T cell proliferation very early after the initiation of the rush. Objective In order to see whether this hyporeactivity was associated with a down regulation of accessory molecules, we studied CD28 surface expression on T lymphocytes from 10 non-atopic controls and from 10 non-atopic patients undergoing rush venom immunotherapy. Methods Peripheral blood samples were collected before the rush (day 0), at the end of the rush (day 1), at day 15 and at day 45. CD28 expression was analysed using flow cytometry with double labelling of the CD4⁺ and CD8⁺ lymphocyte subpopulations. Results At baseline CD28 was expressed at a higher level on T lymphocytes from allergic patients than from control subjects (P < 0.04), and in particular on the CD8 subset (P < 0.01), reflecting a decrease in the suppressive CD8⁺CD28^– subpopulation. No changes were found in the percentages of total CD28⁺ T cells, CD4⁺ CD28⁺ or CD8⁺CD28⁺ cells at the different time points after the initiation of immunotherapy. Conclusion These results suggest that the CD28 pathway is probably involved in the development of allergic reactions, but at least at the phenotypic level, CD28 expression remained unchanged after rush venom immunotherapy.     

Interleukin-6 and tumour necrosis factor alpha are expressed by keratinocytes but not by Langerhans cells.

The presence of human cytokines was examined in parallel skin biopsies and epidermal single cell preparations obtained from normal individuals. Using biotin-avidin-peroxidase and immunofluorescence techniques and antibodies against recombinant cytokines, a granular intercellular/membrane-associated staining for interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha), but not IL-1 alpha or beta, was observed. An epidermal cytoplasmic staining pattern was also detected, which was most pronounced using the anti-rIL-6 antiserum. In the epidermal single cell preparations, membrane-associated staining was detected for both IL-6 and TNF alpha. Double staining revealed that CD1-positive Langerhans cells (LC) failed to express any of the examined cytokines. In vitro binding of rIL-6 or rTNF alpha to skin sections and epidermal single cell preparations indicated that the cell surface-associated IL-6 and TNF alpha originally demonstrated on keratinocytes were truly membrane-bound. Finally, co-cultivation of epidermal cells with an IL-6 responsive cell line, B9, and testing of epidermal cell supernatants in this assay, indicated that the in vivo membrane-bound IL-6 had biological activity.     

Allergen-reactive antibodies are found in nasal fluids from patients with birch pollen-induced intermittent allergic rhinitis,but not in healthy controls

BACKGROUND: Increased levels of allergen-reactive immunoglobulins (Igs) have been reported in nasal fluids from patients with intermittent allergic rhinitis (IAR) sensitive to ragweed and grass. The aims of this study were to make a detailed characterization of nasal fluid Igs in birch pollen-induced IAR. METHODS: Nasal fluids were obtained from 23 patients with birch pollen-induced IAR during and after the birch pollen season, and from 20 healthy controls. Nasal fluid total and Bet v 1-reactive (IgA), IgE and IgG as well as albumin were analyzed by immunoassays. The integrity of IgA and IgG, and the molecular form of IgA were assessed by Western blotting and column fractionation, respectively. RESULTS: Nasal fluid total IgE and IgG, but not IgA, were higher in patients compared with controls. Western blotting indicated no significant degradation of IgA (including S-IgA) and IgG. Most of the IgA, including Bet v 1-reactive antibodies, was of the secretory form and of the IgA1 subclass. Bet v 1-reactive IgA and IgG were present in all patients, but was mostly nondetectable in controls. No significant differences in the levels of Bet v 1-reactive IgA and IgG were found in patients during the birch pollen season compared with off season. Both Bet v 1 and Bet v 2-reactive IgE were nondetectable in most samples. CONCLUSIONS: Nasal fluid Bet v 1-reactive IgA and IgG were found in all patients with birch pollen-induced IAR, but not in controls. However, no significant differences were found between patients during and after the birch pollen season.     

Acquired, but not innate, immune responses to Streptococcus pneumoniae are compromised by neutralization of CD40L

Streptococcus pneumoniae is a significant pathogen of young children and the elderly. Systemic infection by pneumococci is a complex process involving several bacterial and host factors. We have investigated the role of CD40L in host defense against pneumococcal infection. Treatment of mice with MR-1 antibody (anti-CD154/CD40L) markedly reduced antibody responses to the pneumococcal protein PspA, elicited by immunization of purified protein or whole bacteria. In mice immunized with whole bacteria, MR-1 treatment reduced antibody responses to capsular polysaccharides but not cell wall polysaccharides. MR-1 did not suppress antibody responses to isolated capsular polysaccharides but did reduce the production of antibody to a capsular polysaccharide-protein conjugate, indicating that when presented in the context of whole bacteria, the humoral response to capsular polysaccharides is partially T-cell dependent. Despite the reduction of the protective humoral responses to pneumococcal infection, administration of MR-1 had no effect on sepsis, lung infection, or nasal carriage in nonimmune mice inoculated with virulent pneumococci. Thus, short-term neutralization of CD40L does not compromise innate host defenses against pneumococcal invasion.     

Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells.

Priming of CTL by means of heat shock proteins (hsp) is dependent on antigen-presenting cells (APC), which present the hsp-associated peptides, via their cell surface MHC class I molecules, toCD8(+) T cells. It has not yet been established how human (hu) hsp70 interacts with the major (hu)APC, the dendritic cells (DC). Here we show that (hu)hsp70 is specifically internalized intoCD14(-), Toll-like receptor 4(-) monocyte-derived (hu)DC by receptor-mediated endocytosis. We further demonstrate that (hu)hsp70 and (hu)hsp60 share the same receptors on (hu)monocyte-derived DC. Both molecules as well as MHC class I molecules are spontaneously internalized and reach the MHC class II-enriched compartments. Finally, freshly isolated (hu) epidermal Langerhans cells (LC), the DC of the skin, as well as CD34(+)-derived LC do not bind hsp60 or hsp70. Given the likely importance of the internalization of hsp70 by APC in the induction of the immune responses, the finding that hsp60 and hsp70 are internalized through the same receptor(s) may explain why microbial hsp60 represents a major T cell antigen. This may rationalize the use of microbial hsp60 to prime immune responses against microbes. The lack of hsp60/70 receptors on epidermal LC raises the crucial question as to whether absence of priming of the skin and mucosal immune systems by hsp-polypeptide complexes could account for some tissue-specific diseases. This work also points to a potential advantage of using monocyte-derived DC in human immunotherapeutic applications of hsp60/70.     

Functional studies on purified eosinophils and neutrophils from patients with Schistosoma mansoni infections.

Unpurified peripheral blood leucocytes or purified eosinophils and neutrophils from patients with schistosomiasis and from normal individuals were compared for their ability to interact with antibody coated schistosomula of Schistosoma mansoni. There was no difference in the ability of buffy coat cells or neutrophils from patients and from normal individuals to mediate antibody-dependent 51Cr release from labelled schistosomula. However, eosinophils from patients were significantly better than those from normal individuals in causing antibody-dependent 51Cr release. This enhanced activity of eosinophils from patients with schistosomiasis was found to correlate with the intensity of their infection as judged by faecal egg counts. Eosinophils from patients also contained a higher proportion of cells with detectable Fc receptors than those from normal individuals. It is suggested that the difference in the behaviour of eosinophils from patients and from normals may reflect an 'activated' state of these cells in the infected individuals.     

Allergen-specific IL-10-secreting type I T regulatory cells,but not CD4CD25Foxp3 T cells,are decreased in peripheral blood of patients with persistent allergic rhinitis

We investigate the frequencies of CD4⁺CD25⁺Foxp3⁺ T cells and allergen-specific IL-10⁺IL-4^-, IFN-γ⁺IL-4^-, IL-4⁺IFN-γ^-CD4⁺ T cells (which display characteristics of nTreg, Tr1-, Th1- and Th2- cells, respectively) in peripheral blood mononuclear cells (PBMCs) of patients with AR and of healthy individuals. In addition, we estimated the suppressive effect of CD4⁺CD25⁺ Treg cells and allergen-specific, IL-10-secreting cells from both two groups. The frequency of CD4⁺CD25⁺Foxp3⁺ T cells is similar in 43 AR patients compared with 38 healthy subjects. CD4⁺CD25^high cells retain suppressive activity on allergen-stimulated cell proliferation and cytokine production of Th1 but not Th2 cells in both groups. However, the frequency of allergen-specific IL-10⁺IL-4^-CD4⁺ T cells is reduced in AR patients, and correlates inversely to clinical symptom scores. Allergen-specific, IL-10-secreting cells potently suppressed D. pteronyssinus major allergen 1-stimulated cell proliferation and cytokine production (IFN-γ and IL-4) in healthy individuals. Altogether our data indicate that the number and function of CD4⁺CD25⁺ Treg cells from allergic patients are not impaired. However, the deficiency of allergen-specific Tr1 cells may play a role in the development of AR.     

Recombinant Leishmania Hsp90 and Hsp70 are recognized by sera from visceral leishmaniasis patients but not Chagas'' disease patients.

Approximately 70% of the cDNA clones identified by immunoscreening Leishmania donovani expression libraries with serum from a patient with visceral leishmaniasis (kala-azar) were found to encode the highly conserved Hsp90 and Hsp70 members of the heat shock protein family. Recombinant fusion proteins containing the C-terminal portions of L. donovani Hsp90 and Hsp70 were used as target antigens in enzyme-linked immunosorbent assays of various sera. Sera from four patients with visceral leishmaniasis recognized recombinant Leishmania Hsp90 and Hsp70, while sera from seven patients with Chagas' disease did not, despite the fact that Trypanosoma cruzi Hsp90 and Hsp70 share more than 80% amino acid identity with their counterparts in Leishmania spp. Thus, Leishmania Hsp90 and Hsp70 elicit strong humoral responses and are potential candidates for specific serodiagnostic assays capable of distinguishing between L. donovani and T. cruzi infections.     

IL-2 enhances allergic airway responses in rats by increased inflammation but not through increased synthesis of cysteinyl leukotrienes.

BACKGROUND: IL-2 has been shown to increase allergic airway responses in rats. OBJECTIVE: The purpose of this study was to investigate whether induction of inflammation and enhancement of cysteinyl-leukotriene (cys-LT) synthesis were involved in the augmentation of airway responses caused by IL-2. METHODS: Brown Norway rats received human recombinant IL-2 or saline subcutaneously twice a day from day 9 to day 14 after sensitization to ovalbumin (OVA). On day 14, rats underwent either lung lavage or were challenged with an aerosol spray of OVA, the airway responses and biliary excretion of cys-LTs were measured for a period of 8 hours after challenge, and the lung leukocyte numbers were determined after enzymatic digestion of lung tissues. RESULTS: The early response after OVA increased from 184.2% +/- 13.5% in the animals receiving saline (n = 10) to 309% +/- 51% (baseline lung resistance) in IL-2-pretreated animals (n = 17; P <.05). The late response also increased from 19.6 +/- 4.5 (area under the curve of baseline lung resistance vs time) in the animals receiving saline to 37 +/- 5.4 after administration of IL-2 (P <.05). However, IL-2-treated animals had lower levels of biliary cys-LTs during the late response than saline-treated animals but similar levels during the early response. This difference could not be attributed to an increase in LT metabolism, which we assessed by the recovery of 3H-LTC4 instilled intratracheally in challenged or unchallenged rats. When compared with control animals, pretreatment with IL-2 increased all cell types retrieved from lung lavage fluid before OVA challenge (P <.05). After OVA challenge, the total cell yield from lung lavage fluid was also increased, mostly because of an increase in neutrophils (P <.05). Eosinophils and lymphocytes were greater in the lungs of IL-2-treated than vehicle-treated and OVA-challenged rats (P <.01), and IL-2-treated rats had a lower CD4(+)/CD8(+) ratio in the blood after challenge (P <.001). CONCLUSION: In conclusion, IL-2 increases early and late responses in rats, and it induces lung inflammation. Altered airway responses are not attributable to an increase in cys-LT production.     

Autoantibodies to beta 2-adrenergic receptors with antiadrenergic activity from patients with allergic asthma.

The serum gamma-globulin fraction from patients with allergic bronchial asthma (8/8), in contrast to the fraction from nonatopic control subjects (10/10), was found to inhibit the positive chronotropic action of the beta 2-selective adrenergic agonist, clenbuterol, on pyruvate- or lactate-treated cultured neonatal rat heart myocytes. No inhibition was exerted on the positive chronotropic response to prenalterol, which acted via beta 1-adrenergic receptors. The inhibitory effect of the asthmatic gamma-globulins was concentration dependent. It could nearly be abolished by immunoprecipitation with antihuman gamma-globulin and antihuman IgG, but not with antihuman IgM. This finding means that the inhibitory immunoglobulins of the patients with asthma were chiefly autoantibodies of the IgG isotype, capable of cross-reacting with chronotropic beta 2-adrenergic receptors on the cultured rat cardiomyocytes. These findings provide support for the notion that autoimmunity plays a role in the impairment of beta-adrenergic responsiveness in patients with allergic bronchial asthma.     

Live but not heat-killed mycobacteria cause rapid chemotaxis of large numbers of eosinophils in vivo and are ingested by the attracted granulocytes.

We studied leukocyte chemotaxis triggered by a local injection of mycobacteria (Mycobacterium avium and M. smegmatis) in BALB/c and C57BL/6 mice. Our experimental model consisted of the induction of a subcutaneous air pouch in the dorsal area of mice and inoculation 6 days later of 10(8) CFU of myocobacteria. Inflammatory exudates were harvested from the air pouch cavities 15, 30, and 45 min after the injection of the inocula. Injection of the microorganisms resulted in the migration of an elevated number of eosinophilic granulocytes into the inflammatory cavities. At 30 min after the inoculation of the mycobacteria, the air pouches contained between (3.9 +/- 0.3) x 10(5) (M. avium) and (3.3 +/- 0.3) x 10(5) (M. smegmatis) eosinophils, corresponding to more than one-third (41.4 to 38.3%) of the leukocytes present in the inflammatory cavities. Less than one-half of the eosinophils were attracted to the air pouches when the same number of heat-killed mycobacteria were inoculated [(1.3 +/- 0.2) x 10(5) cells for M. avium and (1.5 +/- 0.2) x 10(5) cells for M. smegmatis]. Injection of gram-negative bacteria (Escherichia coli), of latex beads, or of casein resulted in the attraction of inflammatory eosinophils in numbers that were comparable to those attracted by the heat-killed mycobacteria. Our data document the fact that live mycobacteria exert a rapid chemotactic effect on eosinophils. We therefore postulate that mycobacteria either contain or induce the production of an eosinophilotactic factor. Because this chemotactic effect occurs during the acute inflammatory response to mycobacteria, it cannot be due to the formation of immune complexes (a major infection-associated chemotactic factor for eosinophils). The attracted eosinophils had an important role in the local phagocytosis of mycobacteria, as indicated by our finding, derived from thin-section electron microscopy quantifications, that at 30 min after M. avium inoculation the inflammatory exudates contained (2.2 +/- 0.5) x 10(5) mycobacterium-bearing eosinophils (corresponding to 57% of the total eosinophils), as compared with (2.1 +/- 0.1) x 10(5) neutrophils and (1.5 +/- 0.2) x 10(5) macrophages with ingested bacilli. We conclude that mycobacteria induce the attraction of eosinophils to inflammatory sites and that these granulocytes have the capacity to phagocytize these bacilli in situ.     

Mycoplasma fermentans,but not M penetrans,detected by PCR assays in synovium from patients with rheumatoid arthritis and other rheumatic disorders.

AIM/BACKGROUND: Mycoplasmas, especially Mycoplasma fermentans, were suggested more than 20 years ago as a possible cause of rheumatoid arthritis but this hypothesis was never substantiated. In view of the superior sensitivity of the polymerase chain reaction (PCR) assay over culture, the aim was to use this method to seek M fermentans and M penetrans in synovial samples from patients with various arthritides. METHODS: Synovial fluid samples (n = 154) and synovial biopsy specimens (n = 20) from 133 patients with various rheumatic disorders were stored at -80 degrees C for between one and 40 months. Aliquots (500 microliters) of the synovial fluid samples were centrifuged and the deposit, and also the synovial biopsy specimens (approximately 1 g) were placed in lysis buffer with proteinase K for DNA extraction. The DNA was tested by using a semi-nested PCR assay for M fermentans and a single-round PCR for M penetrans. RESULTS: M fermentans was detected in the joints of eight (21%) of 38 patients with rheumatoid arthritis, two (20%) of 10 patients with spondyloarthropathy with peripheral arthritis, one (20%) of five patients with psoriatic arthritis, and four (13%) of 31 patients with unclassified arthritis. M fermentans was not found in the joints of the seven patients with reactive arthritis, the 29 with osteoarthritis or post-traumatic hydrarthrosis, the nine with gouty arthritis, nor the four with chronic juvenile arthritis. M penetrans was not detected in any sample. CONCLUSIONS: These findings show that the presence of M fermentans in the joint is associated with inflammatory rheumatic disorders of unknown cause, including rheumatoid arthritis. However, whether this organism triggers or perpetuates disease of behaves as a passenger remains conjectural.     

Bone morphogenetic protein receptors and activin receptors are highly expressed in ossified ligament tissues of patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is a pathological ossification in the spinal ligament, with formation of ectopic bone mainly through endochondral ossification. Bone morphogenetic proteins (BMPs) and activins are multifunctional proteins that belong to the transforming growth factor-beta superfamily and that have been implicated in the formation of new bone and cartilage. BMPs and activins signal via type I and type II receptors for BMPs (BMPRs) and activins (ActRs), respectively. OP-1/BMP-7 binds to BMPR-II and ActR-II and forms complexes with BMPR-IA and -IB and ActR-I. We studied the expression of BMPR-IA, -IB, and -II, ActR-I, ActR-II, and OP-1/BMP-7 by immunohistochemistry in ossified ligament tissues of patients with OPLL and control ligament tissues from patients with cervical disc herniation. The expression of BMPRs and ActRs was elevated in OPLL compared with controls. Expressions of BMPR-IA, -IB, and -II were observed not only in chondrocytes at the fibrocartilage tissue around the calcified zone but also in fibroblast-like spindle cells at the nonossified ligament. ActR-I and -II were found co-localized in the hypertrophic chondrocytes near the calcified zone and in the ossified tissue. OP-1/BMP-7 was expressed in chondrocytes near the calcified zone. In the control cases, the BMPRs and ActRs were only weakly expressed in the fibrocartilage tissue at the site of ligament attachments to bone and OP-1/BMP-7 was not detected. Enhanced expression of BMPRs at the nonossified ligament in OPLL patients suggests that these cells have a greater potential to differentiate into osteogenic cells than ligament cells from non-OPLL patients. The high expression of BMPRs and ActRs in the ectopic ossified ligament suggests that BMPs and activin may be tightly involved in the pathological ossification process of OPLL.     

Antibodies against Trypanosoma cruzi alkaline antigens are elicited in sera from acute but not chronic human chagasic patients.

The aim of this work was to study the antibody response of acute and chronic chagasic patients against a Trypanosoma cruzi alkaline fraction (FI) in comparison with the reactivity against a T. cruzi acidic antigen, the main cystein proteinase of the parasite named cruzipain, and "natural" antigens. FI-specific antibodies were detected only during the acute phase of the infection and IgM was the main isotype produced, whereas cruzipain-specific antibodies were detected during all phases of the infection. By means of immunoblot and sequencing analysis we identified a 47-kDa FI proteic band recognized by IgM from acute chagasic patients as the T. cruzi glutamate dehydrogenase (GluDH). Furthermore, the antibody response against isolated GluDH showed similar characteristics as the one against FI. We also observed a strict association between the reactivity of IgM against FI and GluDH and IgM natural antibodies. However, reactivity against these alkaline antigens was not modified after absorption of natural antibodies in sera from acute chagasic patients, indicating that these parasite antigens are not recognized by the polyspecific natural antibodies. The most important goal of this report is that for the first time the T. cruzi antigen isoelectric point has been associated with its ability to trigger immunological memory, raising a novel antigen property that should be considered in the selection of antigens used in Chagas' disease diagnostic test and in the design of a vaccine against T. cruzi infection.     

Expression of membrane-bound CD23 in nasal mucosal B cells from patients with perennial allergic rhinitis.

BACKGROUND: CD23 is the low-affinity receptor for IgE on B cells and is thought to play an important role in regulation of IgE production. OBJECTIVE: To measure the expression of membrane-bound CD23 in nasal B cells and examine its correlation with CD4 subtypes or serum IgE levels in patients with perennial allergic rhinitis. METHOD: We used flow cytometric analysis with double, direct immunofluorescence staining of the mucosal-infiltrating lymphocytes to examine the expression of CD23 in nasal mucosal B cells of patients with perennial allergic rhinitis. The expression of CD23 in nasal B cells of patients with nonatopic rhinosinusitis served as a control. RESULT: The ratio of CD23+ B cells to total B cells in patients with perennial allergic rhinitis was significantly higher than in nonatopic controls, whereas that of B cells to total lymphocytes was unchanged. The ratio of CCR4+ CD4 cells to total CD4 cells in allergic patients was significantly higher than in nonatopic controls, whereas the ratio of CXCR3+ CD4 cells to total CD4 cells was unchanged. There was no significant correlation between the percentages of CD23+ B cells and CCR4+ CD4 cells. In addition, the percentage of CD23+ B cells did not correlate with the total IgE level or with the specific IgE level. CONCLUSIONS: Our results indicate that nasal mucosal CD23-bearing B cells, as well as T(H)2 cells, increase in patients with perennial allergic rhinitis. However, the expression of CD23 did not directly correlate with the number of T(H)2 cells in the nasal mucosa.