MALDI-TOF MS analysis of labile Lolium perenne major allergens in mixes.

BACKGROUND: It is well known that allergen extracts used for specific therapy of allergic disorders are commonly stored as mixtures, causing an alteration of its stability. OBJECTIVE: The aim of this report is to identify pollen allergens susceptible to degradation during storage of mixtures containing different sources of proteases in the absence of glycerol as a preserving agent. METHODS: Mixes containing Lolium perenne (Lol p) pollen extract with either Aspergillus fumigatus or Periplaneta americana extracts were prepared and co-incubated for 90 days at 4 degrees C. Samples were taken off at fixed times and comparatively tested by in vitro and in vivo assays with atopic patients. Selected pollinic allergens were subjected to MALDI-TOF MS analysis. RESULTS: ELISA inhibition evidenced the loss of potency from ryegrass extract, and immunoblotting assays showed the degradation of specific pollinic allergens during storage of mixtures containing protease-rich sources. An in vivo intradermal skin assay confirmed the gradual loss of the biological activity of L. perenne pollen extract co-incubated with non-related protease-rich extracts in comparison with that of the control pollen extract. MALDI-TOF MS analysis allowed us to determine that Lol p 1 and Lol p 5 are susceptible to proteolysis whereas Lol p 4 was found to be resistant to degradation during storage. CONCLUSIONS: Lol p 1 and Lol p 5 degradation is responsible for the loss of the biological activity of L. perenne pollen extract when co-incubated with protease-rich fungal and cockroach extracts in the same vial for months in the absence of glycerol as a preserving agent. The integrity of these major allergens must be preserved to increase the vaccine stability and to assure efficacy when mixes are used for immunotherapy.

Proteinase and gelatinolytic activities of house dust mite and cockroach extracts.

The presence of gelatinolytic activity in dust mite and Periplaneta americana allergenic crude extracts were studied. The former presented major activity in a broad band between 45 and 66 kDa and minor activity at 32 kDa, while the latter showed a more complex pattern with gelatinolytic activity at 90, 78, 65, 34, 32 and 24 kDa. When the proteolytic activity patterns of dust mites and cockroach crude extracts were analyzed at three different pH levels, the proteases in both cases were optimally active at pH 6, showed no activity at pH 3.5 and little activity at pH 8.5. The susceptibility of both extracts to a set of well-known protease inhibitors suggested that they are composed of cysteine and serine proteinases, the latter probably being a trypsin-like type. When immunochemical properties were studied, dust mite bands of about 200, 110, 65, 60 and 43 kDa showed immunoreactivity against a polyclonal human anti-dust mite serum, with the band of approximately 200 kDa presenting the highest antigenicity. A similar analysis was applied to the cockroach extract, which exhibited immunoreactive bands at 90, 78, 65 and 34 kDa when incubated with a polyclonal rabbit anti-Blatta serum. Only those of 90, 78 and 65 kDa reacted against a polyclonal human anti-Blatta serum. These results suggested a correlation between some proteases with gelatinolytic activity and the allergenicity of both extracts.

Intestinal infection with Isospora belli.

A review article discussing the taxonomy, parasite life cycle and laboratory diagnosis of Isospora belli is presented. The pathology, epidemiology, clinical findings and therapy of human intestinal infection with Isospora belli are also addressed.

Sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) and extranodal involvement of the orbit.

We present the case of a 5-year-old boy who was referred to the Neurosurgical Service of the San Jorge Children's Hospital for evaluation of right eye proptosis. At the age of one year the diagnosis of sinus histiocytosis with massive lymphadenopathy (SHML) had been rendered on a cervical lymph node biopsy. The right orbit mass causing the proptosis was the result of extranodal involvement of the orbital tissue by SHML. The clinical and pathologic features of this entity are discussed.

A citrus fruit-exclusion diet in sensitive patients and its influence on specific antibodies.

The presence and kinetics of specific IgE and IgG antibodies against the oranges Citrus aurantium sinensis (CAS) and Citrus silension (CS) were studied in 41 atopic and 20 non-atopic children aged 8-12 years. Diagnostic procedures such as intracutaneous skin tests, IgE PRIST and anti-CAS and anti-CS IgE and IgG RAST were performed in both groups. A citrus fruit-exclusion diet was maintained for 180 days. The comparison of the results before and after the diet showed no significant changes in skin reactivity and RAST values, and even a slight increase was recorded in the latter. We have attempted to explain these puzzling findings.

Bone tumors of mixed origin: osteo-liposarcoma and osteo-rhabdomyosarcoma.

1. A complete perusal of the literature revealed twenty cases of primary liposarcoma of bone acceptable as such to the authors. These were tabulated as to location and age. 2. Eight cases of osteo-liposarcoma, primary in bone, were encountered in the literature and an additional case was reported by the authors. 3. The authors described for the first time in the literature a new primary tumor of bone of mixed origin: osteo-rhabdomyosarcoma. After careful perusal of the literature they added three additional cases: two cases, previously reported as primary rhabdomyosarcoma of bone, which on careful evaluation of the radiographs in said publications and the paucity of microphotographs they considered to be osteo-rhabdomyosarcomas, and the other case, previously reported as malignant mesenchymoma of the sternum following radiotherapy for breast cancer. 4. The authors prefer to classify these tumors (osteo-liposarcoma and osteo-rhabdomyosarcoma) as "Tumors of Mixed Origin" and not as "Malignant Mesenchymomas". 5. A complete review of the literature revealed 219 reported "dedifferentiated" chondrosarcomas, or chondrosarcomas "with additional mesenchymal component", among which only nine (9) contained a bona fide rhabdomyosarcomatous component. The rest exhibited other mesenchymal tumors as osteogenic sarcoma, fibrosarcoma, malignant fibrous histiocytoma, angiosarcoma, and undifferentiated sarcoma. The authors recommend to continue classifying these tumors as chondrosarcomas with additional mesenchymal component or even as "dedifferentiated" chondrosarcomas but not as malignant mesenchymomas.

Extraskeletal chondromas.

1. Extraskeletal chondromas occur in three variants: (a) as multiple nodules of synovial chondromatosis within a joint, (b) as a solitary lesion in association with articulations, within or adjacent to them, and (c) as an isolated cartilagenous lesion in the soft tissues, mostly of the hands and feet. 2. There are no histological characteristics which could differentiate articular, para-articular and soft tissue chondromas among themselves. 3. The latter two groups of extraskeletal chondromas are rare and frequently exhibit areas of immature cartilage with worrisome histologic features which could mislead the pathologist to an overdiagnosis of chondrosarcoma. 4. As a general rule, no matter how worrisome the histologic appearance of an extraskeletal, well delimited cartilagenous tumor may be, metastasis have not been recorded. Local recurrences are not infrequent after inadequate surgical removal; however, the tumor can always be controlled by adequate wide re-exision or resection. 5. Two cases of extraskeletal chondromas, one para-articular in the knee and the other in the soft tissue of the arm, have been presented with an exhaustive review of the literature on the subject. 6. It is of interest to note that our para-articular chondroma represents the first case reported in the literature where computerized tomography was utilized in the diagnosis. 7. The location of a soft tissue chondroma in the upper arm is extremely rare and only one previous case has been reported in said location.

Induction of anti-orange antibodies in rabbits.

The capacity of certain foods to cause allergic reactions is well known. The four types of mechanisms that Gell and Coombs described in 1968, are involved in these reactions, although the reaginic antibodies retain the paramount attention of the immunologists. The physiochemical composition of the allergen molecule is the goal of investigators with the purpose to clarify the intrinsic kinetics of antibody synthesis. This paper contributes to the conflicting data about orange allergens especially those obtained from Citrus Aurantium Sinensis and Citrus Silension (CAS and CS, respectively). Glycoproteins were separated by gel filtration through a Sephadex G-50 column. A definite protein peak was obtained meanwhile several hexoses appeared throughout the fractionation procedure. These molecules have adequate physiochemical properties that make them able to trigger the immunological response (molecular weight, definite chemical composition and glycoprotein content). Although CAS and CS have a similar chemical composition a slight inverse proportion of proteins and hexoses was demonstrated between the two classes. Molecular weights were different for CAS (51.500) and for CS (37.000) in comparison with well established protein makers. Ouchterlony revealed two precipitin lines in the CAS-anti-CAS system but none in the CS-anti-CS one. The Boyden technique showed a titre of 1/256 in the first case and only of 1/64 in the second of specific anti-orange antibodies. All the eluted fractions gave negative results although they were concentrated ten times by pre-evaporation. This animal model reinforced the statement that after a long and continuous exposure to orange antigens it was possible to develop specific antibodies. It is assumed that this phenomenon happens in atopic children with it's diagnostic and therapeutic importance.