Identification of carboxymethyl (CM)-binding proteins derived from Lolium multiflorum pollen extract and antibody reactivity in Brazilian allergic patients.

Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1 (IgE median absorbance: 1.22; IgG4 median absorbance: 0.68) and S2 (IgE median absorbance: 1.26; IgG4 median absorbance: 0.85) in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4 (IgE: r=0.9567; IgG4: r=0.9229; P<0.0001) levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy.

Giardia duodenalis: kinetics of cyst elimination and the systemic humoral and intestinal secretory immune responses in gerbils (Meriones unguiculatus) experimentally infected.

This study aimed to determine the pre-patent period and to evaluate the kinetics of cyst elimination and the systemic humoral (IgA, IgG(1), IgG(2a), IgM, IgE) and intestinal secretory (IgA) immune responses in gerbils (Meriones unguiculatus) experimentally innoculated with different doses of Giardia duodenalis trophozoites. Forty-eight animals aged 6-8 weeks were used, equally distributed among six groups, five groups innoculated with different doses of trophozoites (10(1), 10(2), 10(3), 10(4), 10(5)) and one control (non-infected) group. Coproparasitological examinations were carried out daily up to 91 days after inoculation (d.a.i.) to determine the pre-patent period and the kinetics of cyst elimination. Blood and stool samples were weekly collected for antibody assays. The pre-patent period was observed from the 9 d.a.i. onwards, with intermittent elimination of variable quantities of cysts up to 27 d.a.i.. All infected gerbils, irrespective of the dose received, were able to mount systemic humoral immune responses as evidenced by specific IgM titers from 7 to 28 d.a.i., corresponding to the peak of cyst elimination, followed by high and persistent IgG1 titers. Intestinal secretory responses were also seen with two peaks of fecal IgA titers, corresponding to IgM and IgG1 response peaks, respectively. In conclusion, systemic and intestinal humoral immune responses were related to the control of giardiasis in this experimental model.

Mite-specific immunotherapy using allergen and/or bacterial extracts in atopic patients in Brazil.

OBJECTIVE: This study aimed to evaluate the clinical efficacy and antibody response changes after specific immunotherapy (SIT) using Dermatophagoides pteronyssinus (Dpt) allergens with or without bacterial extracts in Brazilian mite-atopic patients. METHODS: One-hundred patients with allergic rhinitis were selected for a randomized double-blind, placebo-controlled trial and distributed into 4 groups: Dpt (Dpt allergen extract), Dpt+MRB (Dpt allergen plus mixed respiratory bacterial extracts), MRB (MRB extract only) and placebo. Rhinitis symptom and medication scores; skin prick test (SPT) to Dpt extract; and serum immunoglobulin (Ig) E, IgG4, and IgG1 levels to Dpt, Der p 1, and Der p 2 allergens were evaluated before and after a year of treatment. RESULTS: After 1 year, the SPT response was reduced in the Dpt group (P=.03), whereas IgE levels to Der p 2 decreased only in the Dpt (P = .048) and Dpt+MRB (P = .005) groups. IgG4 and IgG1 levels to Dpt and Der p 1 increased in the Dpt group (P < .05), whereas in the Dpt + MRB group the IgG1 level only increased to Dpt (P=.001) and the IgG4 only increased to Der p 1 (P=.049). IgE levels to Dpt decreased only in the MRB (P= .005) and Dpt + MRB (P= .001) groups. Rhinitis symptom and medication scores fell in all groups, including the placebo group (P<.001). CONCLUSIONS: SIT using Dpt extract alone was effective in reducing SPT response and IgE levels to Der p 2 allergen, while bacterial extracts induced decreases in IgE levels to whole Dpt extract. However, only groups receiving Dpt allergen had higher levels of IgG1 and IgG4 to Dpt and Der p 1 after a year of treatment.

Identification of carboxymethyl (CM)-binding proteins derived from Lolium multiflorum pollen extract and antibody reactivity in Brazilian allergic patients

Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1 (IgE median absorbance: 1.22; IgG4 median absorbance: 0.68) and S2 (IgE median absorbance: 1.26; IgG4 median absorbance: 0.85) in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4 (IgE: r=0.9567; IgG4: r=0.9229; P<0.0001) levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy.

Taxis but not private cars are mite allergen reservoirs in Brazil.

Indoor allergens are major causative agents in allergic disease development. Besides homes, public transport vehicles have been considered important mite and pet allergen reservoirs. Our recent studies on allergen exposure in automobiles showed that different allergen levels are found in private cars versus taxis. We quantified group 1 Dermatophagoides spp. (Der 1), Felis domesticus (Fel d 1), and Canis familiaris (Can f 1) allergen levels by ELISA in dust samples from 60 taxi and 60 private car upholstered seats. Mean levels of Der 1 and Fel d 1 were significantly higher in taxis than private cars. A significantly higher percentage of taxis (42%) harboring sensitizing levels of Der 1 compared to private cars (5%) was also found. In spite of the low mean Fel d 1 levels, comparison of the percentage of vehicles with moderate Fel d 1 levels showed a significant difference between taxis and private cars (43% vs. 20%). On the other hand, mean Can f 1 levels were significantly higher in private cars compared to taxis concomitant with a significantly higher percentage of private cars containing moderate Can f 1 levels than taxis (53% vs. 28%). We conclude that upholstered seats from Brazilian taxis but not private cars constitute an important mite allergen reservoir. Thus, additional effective measures for the reduction of allergen exposure in vehicles within the global allergen avoidance strategy should also be routinely accomplished to minimize the induction of sensitization and symptoms in allergic patients.

Differential IgE reactivity to Der p 1 and Der p 2 allergens of Dermatophagoides pteronyssinus in mite-sensitized patients.

Several studies have shown that the presence of IgE antibodies to house dust mites (HDM), particularly Dermatophagoides pteronyssinus (Dpt), is an important risk factor for asthma. Allergen immunotherapy is indicated for patients with IgE antibodies to clinically relevant allergens. The aims of this study were to analyze the levels of specific serum IgE to Der p 1 and Der p 2 allergens in mite-sensitized atopic patients and to compare them with both in vivo (skin prick test) and in vitro (IgE-ELISA) sensitizations to Dpt crude extract. Forty-seven atopic patients with allergic rhinitis with or without intermittent or persistent mild asthma and positive skin prick test (SPT) to Dpt total extract were studied. Thirty age-matched healthy subjects with negative SPT to HDM were included as controls. Levels of total IgE and Dpt-, Der p 1- and Der p 2-specific IgE were measured by ELISAs in SPT-positive atopic patients and SPT-negative control subjects. Among 47 symptomatic atopic patients, 27 (57.4%) were double positive IgE to Der p 1 and Der p 2 allergens, 3 (6.4%) were single positive IgE to Der p 1, 4 (8.5%) were single positive IgE to Der p 2, and 13 (27.6%) were double negative IgE to both allergens. There was a significant correlation between Der p 1- and Der p 2-specific IgE levels, but not between Der p 1- or Der p 2-IgE levels and SPT results. The double negative IgE patients had the smallest skin test reactions although they showed high mean levels of total serum IgE. Therefore, the knowledge of specific IgE levels to Der p 1 and Der p 2 major allergens might support physicians for indication or follow-up in mite-sensitized patients under allergen-specific immunotherapy. These approaches might be important for obtaining improved safety and efficacy of the current clinical practice of allergen immunotherapy.

Responses of IgE, IgG1, and IgG4 to concanavalin A-binding Blomia tropicalis antigens in allergic patients.

Blomia tropicalis (Bt) and Dermatophagoides pteronyssinus (Dp) are the prevalent house dust mites in tropical countries and are associated with allergic diseases. Glycosylated antigens are highly immunogenic and involved in different pathologies. We evaluated the presence of IgE, IgG1, and IgG4 to concanavalin A-binding antigens (Bt-Con-A) isolated from Bt-total extract in sera of allergic and non-allergic subjects. Bt-total and Bt-Con-A extracts were evaluated by SDS-PAGE and ELISA for reacting with IgE, IgG1, and IgG4 in sera of 121 patients with allergic rhinitis and 36 non-allergic individuals. All subjects were skin prick tested with Bt-total extract and inhibition tests were performed for IgE, IgG1, and IgG4 using both extracts (Bt-total and Bt-Con-A). Skin prick test showed that 58% of the patients were sensitized to Bt (Bt+), with 52% reactive to both mites (Bt and Dp) and 6% to Bt only. A broad spectrum of proteins (14-152 kDa) was visualized in Bt-total and components >27 kDa for the Bt-Con-A extract. ELISA showed a similar profile of IgE, IgG1 and IgG4 levels in response to Bt-total and Bt-Con-A extracts in different groups, although Bt+ patients showed a lower IgG4 reactivity to Bt-Con-A extract. Specific IgG1 levels were higher in Bt+ patients than in control subjects, and IgG4 levels showed no significant difference among groups. ELISA inhibition showed a partial IgE and total IgG1 and IgG4 cross-reactivity with Dp extract for Bt-total and Bt-Con-A extracts. We conclude that Con-A-binding components isolated from Bt constitute major allergens and are involved in both allergen sensitization (IgE response) and homeostasis maintenance (IgG1 and IgG4 responses).

Functional impact of human collagen alpha2(XI) gene polymorphism in pathogenesis of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is the leading cause of myelopathy in Japan. In earlier studies, we provided genetic linkage and allelic association evidence of distinct differences in the human collagen alpha2(XI) gene (COL11A2) that might constitute inherited predisposition to OPLL. In the present study, a strong allelic association with non-OPLL (p = 0.0003) was observed with an intron 6 polymorphism [intron 6 (-4A)], in which the intron 6 (-4A) allele is more frequently observed in non-OPLL subjects than in OPLL patients. In addition, a newly identified polymorphism in exon 6 [exon 6 (+28A)] was in linkage disequilibrium with the intron 6 (-4A). The functional impact of the polymorphisms was analyzed by comparing the differences in messenger RNA (mRNA) splicing by reverse-transcription polymerase chain reaction (RT-PCR) analysis in cultured cells from the interspinous ligament and an in vitro exon trapping study. The intron 6 (-4A) allele resulted in skipping exon 6 and retaining exon 7, while the exon 6 (+28A) allele was not associated with alteration in mRNA splicing. Similar mRNA species were observed in undifferentiated osteoblast (Ob) cells and in cells from posterior longitudinal ligament of non-OPLL subjects. The region containing exons 6-8 is an acidic subdomain presumably exposed to the surface that could interact with molecules of the extracellular matrix. Accordingly, retaining exon 7 together with removal of exon 6 observed in intron 6 (-4A) could play a protective role in the ectopic ossification process because the same pattern was observed in undifferentiated Ob cells and nonossified posterior longitudinal ligament cells.

Levels of GM-CSF, IL-3, and IL-6 in fluid and tissue from human radicular cysts.

Cytokines released by immune system cells play an important role in cyst enlargement. This study aimed to determine, by ELISA, the levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and IL-6 in fluid and tissue from human radicular cysts. GM-CSF was found in 42.8% of the fluid samples (164.3 pg/mL) and IL-6 in 92.8% (641.4 pg/mL). No IL-3 was detected in any fluid samples. In the tissue samples, 28.6% were positive for IL-3 (369.2 pg/mL), 86.4% for IL-6 (92.4 pg/mL), and 95.8% for GM-CSF (200.5 pg/mL). It can be concluded that GM-CSF and IL-6 were widely found in the fluid and tissue samples. In contrast, IL-3 was found only in the cystic tissue, even though in few lesions. These cytokines may contribute to the inflammation, cystic growth, and bone resorption that characterize cystic lesions.

Comparison of operative results of lumbar disc herniation in manual laborers and athletes.

The results of various surgical treatments for lumbar disc herniation in laborers and athletes were compared on the basis of the relative success of return to work and athletic activity. The rate of successful return to manual labor was the highest at 89% after spinal fusion, but no difference was observed between simple disc excision and percutaneous discectomy. Return to competitive sports was achieved at rate of 81% after percutaneous discectomy, and the time until return to sports activity was significantly shorter than after other procedures. In individuals whose lumbar region is expected to be subjected to great mechanical stress, it is important to take work needs and the special needs of athletes into consideration when selecting the surgical procedure.

Effects of strain distribution in the intervertebral discs on the progression of ossification of the posterior longitudinal ligaments.

STUDY DESIGN: Strain distribution in the intervertebral discs was evaluated biomechanically using an engineering true strain calculation formula. OBJECTIVES: This study was performed to clarify the involvement of dynamic factors in the progression of ossification of the posterior longitudinal ligament. SUMMARY OF BACKGROUND DATA: In patients with ossification of the posterior longitudinal ligament of the cervical spine, ossification frequently progresses after laminectomy. This suggests the involvement of dynamic factors in the progression of ossification. However, these factors have not yet been clarified. METHODS: The analysis was performed on 101 patients with ossification of the posterior longitudinal ligament by employing dynamic lateral x-ray films of the cervical spine. The x-ray films were digitized and used as computer data for calculating the strain distribution. X-ray films were obtained again 5 years later, and the strain distribution and the presence or absence of progression of ossification were evaluated. RESULTS: The progression of ossification of the posterior longitudinal ligament was highly correlated with abnormal strain distribution in the intervertebral discs. Progression of ossification was frequently observed in areas having disc distortion in tension and extension on the posterior longitudinal ligament. CONCLUSIONS: In this study, the area of progression of ossification corresponded to the area showing uneven strain distribution and resultant concentration of dynamic stress. These results suggest an important role for dynamic factors in the progression of ossification of the posterior longitudinal ligament.

Interobserver and intraobserver reliability of the japanese orthopaedic association scoring system for evaluation of cervical compression myelopathy.

STUDY DESIGN: The inter- and intraobserver reliabilities of an assessment scale for cervical compression myelopathy were examined statistically. This scoring system consists of seven categories: motor function of fingers, shoulder and elbow, and lower extremity; sensory function of upper extremity, trunk and lower extremity; and function of the bladder. It evaluates the severity of myelopathy by allocating points based on degree of dysfunction in each category. OBJECTIVES: To determine the inter- and intraobserver reliabilities of the revised scoring system (17 - 2 points) for cervical compression myelopathy proposed by the Japanese Orthopedic Association. SUMMARY OF BACKGROUND DATA: Several scales to assess clinical outcome from treatment of cervical compression myelopathy have been proposed. Most of these scales include items evaluated by observers. However, no system, including the Japanese Orthopedic Association scoring system, has yet been validated in terms of interobserver reliability. METHODS: From five different university hospitals, 10 spine surgery specialists, 10 orthopedic surgeons who had just passed the board examination of the Japanese Orthopedic Association, and 13 residents in the first or second year of orthopedic residency programs were chosen. The participants in this study were 29 patients with myelopathy secondary to ossification of the posterior longitudinal ligament selected from five participating university hospitals. Several surgeons interviewed each patient twice at intervals of 1 to 6 weeks. Inter- and intraobserver reliabilities of the total score for all categories were evaluated by the intraclass correlation coefficient. The extension of the kappa coefficient of Kraemer also was calculated for each category to assess reliability of multivariate categorical data. RESULTS: The interobserver reliability of the total score for the first interview (intraclass correlation coefficient = 0.813) and the intra- and interobserver reliabilities of the total score (intraclass correlation coefficient = 0.826) were high. The level of experience and the hospital slightly affected the reliability of the Japanese Orthopedic Association scoring system. The kappa values for intraobserver data generally were high in each category, whereas the kappa values for interobserver data were relatively low for the categories of shoulder-elbow motor function and lower extremity sensory function. CONCLUSIONS: The inter- and intraobserver reliabilities of the Japanese Orthopedic Association scoring system for cervical myelopathy were high, suggesting that this system is useful for assessment of cervical myelopathy in comparative studies of treatment.

Genetic study of ossification of the posterior longitudinal ligament in the cervical spine with human leukocyte antigen haplotype.

To evaluate the genetic background of ossification of the posterior longitudinal ligament, the relationship between the presence of absence of ossification and human leukocyte antigen haplotypes was studied in 33 families of patients with ossification of the posterior longitudinal ligament. The study revealed that human leukocyte antigen haplotypes formed certain types of clusters, and that some human leukocyte antigen haplotypes were very rare in the Japanese population, suggesting the involvement of human leukocyte antigen-linked factors in the pathogenesis of ossification of the posterior longitudinal ligament of the cervical spine. In the families of these patients, ossification of the posterior longitudinal ligament was demonstrated by radiography in 56% (10/18) of the siblings. Each of these siblings shared both human leukocyte antigen haplotypes with the patient. None of those who shared only one human leukocyte antigen haplotype with the patient had developed ossification of the posterior longitudinal ligament. From these findings, the presence of both pathogenic human leukocyte antigen haplotypes is considered to be necessary for the development of ossification of the posterior longitudinal ligament, and this genetic predisposition may be activated by multiple factors, including regressive degeneration due to aging and the environment.

Restriction fragment length polymorphism of genes of the alpha 2(XI) collagen, bone morphogenetic protein-2, alkaline phosphatase, and tumor necrosis factor-alpha among patients with ossification of posterior longitudinal ligament and controls from the Japanese population.

STUDY DESIGN: The present study analyzed the restriction fragment length polymorphism patterns of alpha 2(XI) collagen, bone morphogenetic protein-2, alkaline phosphatase, and tumor necrosis factor-alpha genes in patients with ossification of the posterior longitudinal ligament. This study investigates the genetic polymorphism of bone-induced factors in patients with ossification of the posterior longitudinal ligament and compares it with healthy control subjects. OBJECTIVES: To clarify the genetic markers linked to ossification of the posterior longitudinal ligament. SUMMARY OF BACKGROUND DATA: Ossification of the posterior longitudinal ligament is a genetic disease associated with abnormal calcium metabolism involving the posterior longitudinal ligament. Previous genetic studies have not identified the pathologic mechanism of ossification of the posterior longitudinal ligament. Histopathologic studies of ossification of the posterior longitudinal ligament and the animal model, the spinal hyperostotic mouse, have revealed an increase in Type XI collagen and bone morphogenetic protein-2 expression. METHODS: Eighteen Japanese patients with ossification of the posterior longitudinal ligament and 51 healthy, unrelated control subjects were investigated for the restriction fragment length polymorphism patterns of COL11A2, bone morphogenetic protein-2, alkaline phosphatase, and tumor necrosis factor-alpha, genes with various restriction endonucleases. RESULTS: The gene frequencies of COL11A2 obtained with BamHl (10.0 kb fragment) and HindIII (19.0 kb fragment) observed in patients with ossification of the posterior longitudinal ligament were higher compared with control subjects (0.43 and 0.14, respectively). These differences were statistically significant (BamHl P = 0.018; Hindlll P = 0.046). Two new restriction fragment length polymorphism patterns were detected of the bone morphogenetic protein-2 gene with Mspl and Taql and one already known restriction fragment length polymorphism pattern of the tumor necrosis factor-alpha gene with Ncol. However, they were not significantly different from the control subjects. CONCLUSIONS: Seven restriction fragment length polymorphisms of COL11A2 gene were identified. Two of them (BamHl, 10.0/10.0 kb genotype; HindIII, 19.0/19.0 kb genotype) were significantly different in patients with ossification of the posterior longitudinal ligament.

Candida albicans allergen immunotherapy in recurrent vaginal candidiasis.

Recurrent vaginal candidiasis is a worldwide problem affecting millions of women. Candida albicans is a potent allergen in some situations and it has been suggested that local hypersensitivity to this yeast can be a factor in the prolongation of the disease. A small number of studies show some benefits of allergen immunotherapy in these patients. We evaluated the efficacy of C. albicans allergen immunotherapy in women with immediate skin test positive for this fungus. We conducted a prospective cohort study of women with recurrent vaginal candidiasis referred by gynecologists to an allergist. All patients had established positive C. albicans in vaginal cultures and were unresponsive to other modes of treatment. None had diabetes or AIDS. In their allergy evaluation, they had an immediate (prick or intradermal) skin test positive to C. albicans. These women were offered the option of C. albicans allergen immunotherapy for a period of 24 months. The efficacy of therapy was evaluated by comparison of the mean incidence of episodes per year before and after immunotherapy Over a period of 33 months, 34 women were enrolled (mean age 33.5 years; range 18-57). They were treated with weekly injections of C. albicans allergenic extract over a mean (+/- SD) period of 17.4+/-7.2 months (4-24 months). Twenty-two of the women had improvement in symptoms. There were complete responses, i.e., absence of acute episodes for 2 years, in nine patients (26%) and partial responses, i.e., decrease in the number and intensity of episodes, in 13 patients (38%). Overall there was a 64% improvement in these patients (95% CI: 46.5-79.9). Eleven patients showed no improvement and one worsened. The effects were evident after 2-12 months of therapy (mean 3.5 months). The mean incidence of episodes of vaginitis per year decreased from a mean (+/- SD) of 8.5+/-2.6 to 3.6+/-4.3 (p = 0.000). The study also showed that the majority of patients were atopic (70%), had allergic rhinitis (67%) and familial history of allergy (70%). Our results suggest that in a subgroup of patients with recurrent vaginal candidiasis, C. albicans allergen immunotherapy lowers the number and intensity of episodes.

Mite allergen levels and acarologic analysis in house dust samples in Uberaba, Brazil.

Mite allergen exposure has been widely related to sensitization and development of allergic diseases. This study intended to evaluate the degree of allergen exposure in Uberaba, Brazil, through the measurements of Der f 1 and Der p 1 allergen levels associated with the acarologic analysis in house dust samples. A total of 240 dust samples were collected from 60 houses through vacuuming sofas and bedding, during the months of March and July 2000. Indoor temperature and relative humidity were also measured. Mites were counted and identified under light microscopy and allergen levels were measured by two-site monoclonal antibody ELISAs. The major mite family was Pyroglyphidae (39.4%), having D. pteronyssinus as the most frequent species (15.6%), followed by D. farinae (12.3%) and E. maynei (7.9%). The family Glycyphagidae was less commonly found (4.8%), with Blomia tropicalis (4.4%) as its majoritary member. The highest levels of Der f 1 and Der p 1 allergens were found in bedding samples in March (31.7 and 0.9 microg/g of dust, respectively), with Der f 1 levels significantly higher than Der p 1 (p < 0.0001). There was a significant positive correlation between the mite number and allergen levels. These results indicate that Dermatophagoides sp are the most frequent mites in our region followed by E. maynei. Therefore, the knowledge of the local mite fauna would improve the means of investigating the association between allergen exposure and sensitization, allowing the addition of new mite extracts in diagnostic tests.

Retinoid and bone metabolic marker in ossification of the posterior longitudinal ligament.

The pathogenesis of ossification of the posterior longitudinal ligament (OPLL) remains to be elucidated, though etiologic factors for OPLL have been identified. High levels of serum retinol and retinol binding protein (RBP) have been observed in patients with diffuse idiopathic skeletal hyperostosis (DISH). OPLL is often associated with DISH. In this study, the levels of serum retinol and RBP were determined in 70 patients with OPLL in the cervical spine, and compared with those in normal subjects. Bone metabolic markers of serum intact osteocalcin, urinary pyridinoline and deoxypyridinoline were examined as well. Among female patients, level of serum RBP was significantly higher in those in their 60's, and those with mixed type OPLL. Level of serum RBP was significantly higher in both sexes, and retinol was exhibited higher in female patients, if they were associated with DISH. Patients with OPLL exhibited no abnormal bone metabolic marker levels. These findings suggest that vitamin A may play a role in the development of OPLL.

Histochemical and immunohistochemical study on the skin of patients with ossification of the posterior longitudinal ligament in the cervical spine.

We present an immunohistochemical study on extracellular matrix (ECM), decorin, in the epidermis and dermis of patients with ossification of the posterior longitudinal ligament (OPLL) to clarify the abnormality in the extracellular matrix of the skin of OPLL. Localization of decorin and TGF-beta in the skin was studied. All patients with OPLL showed a diffuse strong staining of decorin in the entire epidermal layer. The epidermis was also positive for TGF-beta antibody staining. Patients with cervical spondylotic myelopathy showed weak or no staining of decorin. Abnormality in the regulating system of TGF-beta may be involved in the precipitation of extracellular matrix.

Potent stimulation of murine B cells to proliferate and to secrete immunoglobulins by a lipoteichoic acid-like molecule produced by Clostridium botulinum C and D.

Bacterial products have served as important immunological tools to study lymphocyte activation. The lipopolysaccharides of the Gram-negative bacteria are well known to be potent activators of B lymphocytes. Several Gram-positive bacteria produce exotoxins that are superantigens for T cells. In the present study, we demonstrate that the Gram-positive bacteria Clostridium botulinum C and D produce a high molecular weight mitogen (Cb mitogen) that is a potent activator of murine B lymphocytes. The Cb mitogen was discovered as a consequence of our attempt to investigate a possible superantigen activity present in the botulinum exotoxins. We observed initially that mouse spleen cells were strongly stimulated to proliferate by culture supernatants of C. botulinum C and D. However, the characterization of the responding cell ruled out superantigen because only the B lymphocytes were stimulated to proliferate and to secrete immunoglobulins, and they did so independent of T cell help. In addition, the molecular characterization of the Cb mitogen demonstrated that the purified botulinum toxin was devoid of mitogenic activity. In contrast, the fractionation of the culture supernatant of C. botulinum C in an FPLC Superose 12 column indicated that the Cb mitogen was present in the void volume of the column (MW > or = 300 kDa) which had no toxigenic activity. However, the fractions containing molecules of 150 kDa were highly toxic for mice and had no mitogenic activity. The possibility that LPS was present as a contaminant in the Cb mitogen preparations was excluded because spleen cells from the LPS non-responder C3H/HeJ mice responded well to the Cb mitogen, and the antibiotic polymyxin B, which is an inhibitor of LPS, had no effect on the Cb-mitogen activity. However, an anti-lipoteichoic acid monoclonal antibody (3-1 mAb) inhibited to a great extent the proliferation of spleen cells induced by the Cb mitogen but had no effect on the LPS or concanavalin A stimulation of these cells. Moreover, the Cb mitogen was specifically adsorbed and eluted from a protein G Sepharose column to which the anti-lipoteichoic acid 3-1 mAb had been conjugated. These results support the view that lipoteichoic acid is a selective B cell mitogen.

IgE cross-reactivity between Lolium multiflorum and commercial grass pollen allergen extracts in Brazilian patients with pollinosis.

Lolium multiflorum (Lm) grass pollen is the major cause of pollinosis in Southern Brazil. The objectives of this study were to investigate immunodominant components of Lm pollen allergens and the cross-reactivity of IgE with commercial grass pollen allergen extracts. Thirty-eight serum samples from patients with seasonal allergic rhinitis (SAR), 35 serum samples from patients with perennial allergic rhinitis (PAR) and 30 serum samples from non-atopic subjects were analyzed. Allergen sensitization was evaluated using skin prick test and serum IgE levels against Lm pollen extract were determined by ELISA. Inhibition ELISA and immunoblot were used to evaluate the cross-reactivity of IgE between allergens from Lm and commercial grass pollen extracts, including L. perenne (Lp), grass mix I (GI) and II (GII) extracts. IgE antibodies against Lm were detected in 100% of SAR patients and 8.6% of PAR patients. Inhibition ELISA demonstrated IgE cross-reactivity between homologous (Lm) and heterologous (Lp or GII) grass pollen extracts, but not for the GI extract. Fifteen IgE-binding Lm components were detected and immunoblot bands of 26, 28-30, and 32-35 kDa showed >90% recognition. Lm, Lp and GII extracts significantly inhibited IgE binding to the most immunodominant Lm components, particularly the 55 kDa band. The 26 kDa and 90-114 kDa bands presented the lowest amount of heterologous inhibition. We demonstrated that Lm extract contains both Lm-specific and cross-reactive IgE-binding components and therefore it is suitable for measuring quantitative IgE levels for diagnostic and therapeutic purposes in patients with pollinosis sensitized to Lm grass pollen rather than other phylogenetically related grass pollen extracts.