Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen

BACKGROUND: A rapid method for the purification of the major 43-kDa allergen of Cupressus arizonica pollen, Cup a 1, was developed. METHODS: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. RESULTS: Immunochemical analysis of Cup a 1 confirmed that the allergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and allergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients allergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF(3) (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF(3)/GnGXF(3) (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF(3)/Gn(GF)XF(3) (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. CONCLUSION: The rapid purification process of Cup a 1 allowed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.     

Immunohistologic analysis of a human pulmonary alveolar macrophage antigen

PAM1 is a 200-kDa polypeptide antigen present on lavaged human alveolar macrophages but not on monocytes, peritoneal macrophages, breast milk macrophages, or other normal hematopoietic cells studied by flow cytometry. We have characterized the distribution of expression of this antigen by cells in tissues by using immunohistologic techniques. Normal and diseased lung as well as lymph nodes, spleen, kidney, liver, GI tract, and skin were studied. PAM1 was expressed strongly on the surface and weakly in the cytoplasm of most alveolar macrophages in all 15 of the lung specimens. Occasional interstitial macrophages had weak to moderate staining for this antigen but the majority did not stain. The distribution, pattern, and intensity of staining for PAM1 was the same in normal lung specimens and those with interstitial pneumonitis, despite the increase in mononuclear cells in the latter. Dermal histiocytes and Kuppfer cells expressed PAM1 weakly. Sinus histiocytes in lymph nodes were moderately to strongly positive. Although lymphoid cell suspensions (tonsil) were negative by flow cytometry, five of six lymph nodes had positive cells by immunohistology. PAM1 was also detected on endothelial cells of splenic sinusoids in all 6 specimens but not on any other endothelium. Hence, while PAM1 is expressed most strongly on alveolar macrophages, it can also be demonstrated in other locations using sensitive immunohistologic techniques. Since circulating monocytes are antigen negative and some lung interstitial macrophages bear antigen, PAM1 may be a useful marker for studies of the differentiation of mononuclear cells in the lung.     

In vivo activation of NK cells induces inhibition of lung colonization of H-2 positive and H-2 negative fibrosarcoma tumor clones

The role of different tilorone analogs in the abrogation of the metastatic spread of H-2 positive and H-2 negative tumor clones was studied. Pre-treatment of BALB/c mice with RMI 10,874DA compound completely abolished lung colonization of an H-2 negative (GR9.B9) MCA-induced fibrosarcoma clone in an experimental metastasis assay. This effect was also evident when clones were treated with other tilorone analogs (R11,567DA or R11,513DA). Other H-2 positive and H-2 negative chemically induced fibrosarcoma clones were also tested. The effect was not due to direct toxicity of the tilorone analog on tumor cells, but instead was dependent on NK cells; this was suggested by the finding that treatment of mice with anti-asialo GM₁ abrogated the effect of the tilorone analog (RMI 10,874DA compound). Interestingly, the inhibition of lung colonization after intravenous injection was again observed regardless of the H-2 phenotype of the tumor clones, and H-2⁺ and H-2^– clones were similarly inhibited.In vitro assays of NK sensitivity of tumor clones showed that lysis varied depending on the H-2 phenotype of tumor clones, indicating an absence of correlation betweenin vivo andin vitro results.     

Neutropenia induced by platelet-activating factor (PAF-acether) released from neutrophils: The inhibitory effect of prostacyclin (PGI2)

Soluble and phagocytic stimuli released PAF-acether from PMN leucocytes, as determined by chromatography and bioassay by platelet aggregation. The same material caused aggregation of human and rabbit PMN leucocytesin vitro which was inhibited by ETYA and PGI₂. PGI₂ also inhibited PAF-acether release by PMN leucocytes and,in vivo, PGI₂ abolished not only PAF-acether-induced, but also immune complex or C5a-induced thrombocytopenia and neutropenia in rabbits. These data suggest that PAF-acether may be involved in activation of both platelets and PMN leucocytesin vivo.     

The endocannabinoid system in the physiology and pathophysiology of the gastrointestinal tract

Numerous investigations have recently demonstrated the important roles of the endocannabinoid system in the gastrointestinal (GI) tract under physiological and pathophysiological conditions. In the GI tract, cannabinoid type 1 (CB1) receptors are present in neurons of the enteric nervous system and in sensory terminals of vagal and spinal neurons, while cannabinoid type 2 receptors are located in immune cells. Activation of CB1 receptors was shown to modulate several functions in the GI tract, including gastric secretion, gastric emptying and intestinal motility. Under pathophysiological conditions induced experimentally in rodents, the endocannabinoid system conveys protection to the GI tract (e.g. from inflammation and abnormally high gastric and enteric secretions). Such protective activities are largely in agreement with anecdotal reports from folk medicine on the use of Cannabis sativa extracts by subjects suffering from various GI disorders. Thus, the endocannabinoid system may serve as a potentially promising therapeutic target against different GI disorders, including frankly inflammatory bowel diseases (e.g. Crohns disease), functional bowel diseases (e.g. irritable bowel syndrome) and secretion- and motility-related disorders. As stimulation of this modulatory system by CB1 receptor agonists can lead to unwanted psychotropic side effects, an alternative and promising avenue for therapeutic applications resides in the treatment with CB1 receptor agonists that are unable to cross the blood–brain barrier, or with compounds that inhibit the degradation of endogenous ligands (endocannabinoids) of CB1 receptors, hence prolonging the activity of the endocannabinoid system.     

High frequency of altered HLA class I phenotypes in invasive breast carcinomas

We studied 105 tumor samples obtained from patients diagnosed as having breast carcinomas for HLA class I and II (DR) antigen expression, using a panel of mAbs defining HLA-monomorphic, locus-specific and allele-specific determinants. Peripheral blood lymphocytes from patients were also typed for HLA alleles. The results indicated total HLA class I losses in 55 patients (52.3%), HLA-A locus losses in four patients (3.8%), HLA-B locus losses in eight patients (7.6%), and A, B, locus losses in 10 patients (9.5%). The remaining 28 patients whose tissues reacted positively with monomorphic- and locus-specific mAbs were tested for HLA allelic losses using several anti-HLA mAbs defining A2, A3, A9, B8, B12, etc. Of these 28 patients, 16 (57%) showed one or more losses of HLA reactivity. These results indicated that in 88.5% of patients we detected a particular HLA-altered tumor phenotype. The downregulation of HLA class I antigens in breast carcinomas may thus be more frequent than previously reported, and patients without HLA class I downregulation may be the exception rather than the rule. It cannot be ruled out that HLA alterations are present in some of the 12 patients with an apparently normal HLA phenotype, as some HLA alleles could not be studied because of the lack of appropriate mAbs. These HLA alterations could represent an important step associated with tumor invasion, conferring to the tumor cells the ability to escape from T-lymphocyte recognition.     

Psychopathological dimensions of depression: a factor study of the 17-item Hamilton depression rating scale in unipolar depressed outpatients

BACKGROUND: Agreement on the factor structure of the Hamilton Depression Rating Scale (HDRS) has not been consistent among studies, and some investigators argued that the scale's factor structure is not reliable. This study aimed at shedding more light on this debated issue. METHODS: We studied 186 adults with unipolar depression (Major Depressive Disorder, n=80; Dysthymic Disorder, n=71; Depressive Disorder Not Otherwise Specified, n=25; Adjustment Disorder, n=10). They had no comorbid DSM-IV axis I or axis II disorders, and had received no treatment with antidepressant drugs in the previous 2 months. The factor structure of the scale was studied using the principal factor method, followed by oblique rotation. Factor scores were computed for each subject using the regression method. RESULTS: Using the scree-test criterion for factor extraction, we obtained a four-factor solution, explaining 43.8% of total variance. The four factors extracted were identified as (1) somatic anxiety/somatization factor; (2) a psychic anxiety dimension; (3) a pure depressive dimension; and (4) anorexia factor. Patients with Major Depressive Disorder scored significantly higher than patients with other diagnoses on the pure depressive dimension. LIMITATIONS: These results need to be replicated in different cultures, using analogous factoring techniques. CONCLUSIONS: Though not exhibiting factorial invariance in the stricter sense of the term, the 17-item HDRS did exhibit a relatively reliable factor structure. Our analysis provides further evidence that the scale is multidimensional. However, as long as the multidimensional character of the scale is taken into account the scale should be able to play a useful role in clinical research.     

The role of pulmonary CO2 flow in the control of the phase i ventilatory response to exercise in humans

To gain an insight into the origin of the phase I ventilatory response to exercise (ph I) in humans, pulmonary ventilation WE) and end-tidal partial pressures of oxygen and carbon dioxide (P _ETO₂ and P _ETCO₂, respectively) were measured breath-by-breath in six male subjects during constant-intensity exercise on the cycle ergometer at 50, 100 and 150 W, with eupnoeic normocapnia (N) or hyperpnoeic hypocapnia (H) established prior to the exercise test. Cardiac output (Q₂) was also determined beat-by-beat by impedance cardiography on eight subjects during moderate exercise (50 W), and the C0₂ flow to the lungs (Q₂·C_vCO₂ where C_vCO₂ is concentration of CO₂ in mixed veneous blood) was estimated with a time resolution of one breathing cycle. In N, the initial abrupt increase of PE during ph I (V_E approximately 18 l · min^–1 above rest) was followed by a transient fall. When P _ETCO₂ started to increase (and P _ETO₂ decreased) V_E increased again (phase II ventilatory response, ph II). In H, during ph I V_E was similar to that of N. By contrast, during ph II V_E kept gradually decreasing and started to increase only when P _ETCO₂ had returned to approximately 40 mmHg (5.3 kPa). Thus, as a result of the prevailing initial conditions (N or H) a temporal shift of the time-course of V_E during ph II became apparent. No correlation was found between C0₂ flow to the lungs and V_E during ph I. These results are interpreted as suggesting that an increased C0₂ flow to the lungs does not constitute an important factor for the initial hyperventilatory response to exercise. They are rather compatible with a neural origin of ph I, and would support the neurohumoral theory of ventilatory control during exercise.     

Trypanosoma cruzi histone H1 is phosphorylated in a typical cyclin dependent kinase site accordingly to the cell cycle

Histone H1 of most eukaryotes is phosphorylated during the cell cycle progression and seems to play a role in the regulation of chromatin structure, affecting replication and chromosome condensation. In trypanosomatids, histone H1 lacks the globular domain and is shorter when compared with the histone of other eukaryotes. We have previously shown that in Trypanosoma cruzi, the agent of Chagas' disease, histone H1 is phosphorylated and this increases its dissociation from chromatin. Here, we demonstrate using mass spectrometry analysis that T. cruzi histone H1 is only phosphorylated at the serine 12 in the sequence SPKK, a typical cyclin-dependent kinase site. We also found a correlation between the phosphorylation state of histone H1 and the cell cycle. Hydroxyurea and lactacystin, which, respectively, arrest parasites at the G1/S and G2/M stages of the cell cycle, increased the level of histone H1 phosphorylation. Cyclin-dependent kinase-related enzymes TzCRK3, and less intensely the TzCRK1 were able to phosphorylate histone H1 in vitro. Histone H1 dephosphorylation was prevented by treating the parasites with okadaic acid but not with calyculin A. These findings suggest that T. cruzi histone H1 phosphorylation is promoted by cyclin dependent kinases, present during S through G2 phase of the cell cycle, and its dephosphorylation is promoted by specific phosphatases.