Accumulation of surfactant protein D in human pulmonary alveolar proteinosis.

Surfactant protein D (SP-D) is a collagenous calcium-dependent carbohydrate-binding protein that is structurally related to the serum mannose-binding proteins and pulmonary surfactant protein A. SP-D was initially characterized as a biosynthetic product of freshly isolated rat type II cells and first purified in chemical amounts from bronchoalveolar lavage of rats with silica-induced alveolar lipoproteinosis. The present studies describe the characterization of human SP-D isolated from therapeutic bronchoalveolar lavage of patients with pulmonary alveolar proteinosis. Human proteinosis SP-D was extracted from the 10,000 x g pellet of bronchoalveolar lavage with 100 mmol/L glucose or ethylenediamine tetraacetic acid, and specifically bound to and eluted from maltosyl-agarose. The protein cross-reacted with monospecific antibodies to rat SP-D by enzyme-linked immunosorbent assay and immunoblot and eluted near the position of rat SP-D on reverse-phase high performance liquid chromatography. When chromatographed on 4% agarose (A-15M) in the presence of ethylenediamine tetraacetic acid, the solubilized human proteinosis SP-D eluted near the void volume and earlier than rat SP-D dodecamers or human SP-D multimers in the lavage supernatant. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of proteins in the lavage pellet with antibodies to the carbohydrate-binding domain of proteinosis human SP-D demonstrated covalently cross-linked multimers of SP-D monomers (43 kd, reduced) and multimers of trimeric components stabilized by disulfide and non-disulfide bonds. These studies describe the isolation and biochemical characterization of human SP-D and demonstrate the abnormal accumulation of this protein in the air spaces of patients with alveolar proteinosis.     

Preventive and therapeutic effects of oral tolerance in a murine model of asthma

Allergic asthma is an inflammatory disease of the airways, and Th2 cells secreting IL-4 and IL-5 play a pivotal role in its pathogenesis. We have previously demonstrated that oral tolerance can be induced and maintained more profoundly in a Th2-related immune response, and that an ongoing immune response can be suppressed by the oral administration of antigen combined with an appropriate feeding regimen. In the present study, we examined the preventive and therapeutic effects of the oral administration of allergen on a Th2-mediated immune disorder using a murine model of asthma. Our results show that the development of asthma can be blocked completely by orally administering allergen. Airway hyperreactivity, allergen-specific IgE production, Th2-derived cytokines, allergen-induced T cell proliferation and the infiltration of inflammatory effector cells into the lung were prevented by such oral administration. To assess the therapeutic effects of oral administration on the progression of asthma, we tested the effects of oral tolerance in an established asthma model, and found that a multiple high dose-feeding regimen was effective at suppressing the progression of mild asthma. In the high dose-feeding group, the number of eosinophils in bronchoalveolar lavage fluid was reduced and airway reactivity also decreased. However, this was insufficient to reduce airway reactivity and eosinophilia in bronchoalveolar lavage fluid in cases of severe asthma. These results demonstrate that allergic asthma may be ameliorated by feeding allergen; there is hope that these results will provide a new immunotherapeutic strategy for allergic asthma.     

Somatic von hippel-lindau mutation in clear cell papillary cystadenoma of the epididymis

Papillary cystadenoma of the epididymis is an uncommon benign lesion that may occur sporadically or as a manifestation of von Hippel—Lindau (VHL) disease. Neither immunohistochemical studies nor molecular genetic analyses of the VHL gene have been reported previously for this lesion. The authors describe two cases of clear cell papillary cystadenoma of the epididymis, both of which were initially confused with metastatic renal cell carcinoma. Both lesions showed positive immunohistochemical staining for low and intermediate molecular weight keratins (Cam 5.2 and AE1/AE3), EMA, vimentin, α₁-antitrypsin, and α₁-antichymotrypsin. Each was negative for CEA. Because clear cell papillary cystadenoma is similar to renal cell carcinoma histologically, and because both occur as components of the von Hippel—Lindau disease complex, the authors analyzed both cases for the presence of mutations in the VHL gene. A somatic VHL gene mutation was detected in one of the two tumors by polymerase chain reaction followed by single-strand conformation polymorphism analysis. Direct sequencing revealed a cytosine to thymine transition at nucleotide 694, resulting in the replacement of an arginine with a stop codon after the sixth amino acid of exon 3. As the VHL gene is believed to function as a tumor suppressor gene, VHL gene mutations may play a role in the initiation of tumorigenesis in sporadic cystadenomas of the epididymis.     

Modification of collagen matrices for enhancing angiogenesis

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.     

Epstein-Barr virus in gastric carcinomas with lymphoid stroma

AIMS: To clarify the relationship between the Epstein-Barr virus (EBV) and gastric carcinoma with lymphoid stroma (GCLS) in Koreans, and to characterize the EBV-positive GCLS. METHODS AND RESULTS: EBV infection was examined using EBER in-situ hybridization and polymerase chain reaction in 45 cases of GCLS among Koreans, and in 292 consecutive cases of gastric carcinomas without lymphoid stroma (non-GCLS) as controls. EBV infection was found in 30 tumours (67%) of GCLS and 10 tumours (3.4%) of non-GCLS (P < 0.05). EBV-positive GCLS was more prevalent in males, poorly differentiated histological type and diffuse type in Lauren's classification, and tended to be located more in the middle third of the stomach than EBV-negative GCLS (P < 0.05). p53 overexpression was observed in 22% of GCLS (17% of EBV-positive GCLS and 33% of EBV-negative GCLS), and 34% of non-GCLS (EBV-positive GCLS vs. non-GCLS: P = 0.056). The survival of the patient with GCLS was not correlated with EBV infection or p53 immunoexpression (follow-up period: 11-97 months). CONCLUSIONS: GCLS in Koreans is strongly associated with EBV infection. The prognosis in GCLS is not dependent upon either the status of EBV infection or the status of p53 immunoexpression.     

Influence of residual stress on bonding strength and fracture of plasma-sprayed hydroxyapatite coatings on Ti-6Al-4V substrate

Six hydroxyapatite coatings (HACs) were plasma sprayed on Ti-6Al-4V substrates by varying the substrate temperatures and the cooling conditions. This study is aimed not only to measure the residual stress of HACs under various conditions, but also to assess the influence of residual stress in HACs on their bonding strength. The residual stress and bonding strength were measured by XRD "sin2 psi" technique and standard adhesion test (ASTM C-633), respectively. The result of the study clearly established the relationship between bonding strength and residual stress. The arguments leading to the above conclusion were discussed in detail. Fractographic analysis indicated that fracture of the system occurred mainly inside the hydroxyapatite coating under lower residual stress; as residual stress increased, fracture tended to occur more easily along the crucial HA-Ti alloy substrate interface. A mechanism was presented for the relationships between residual stress, fracture behaviour and bonding strength for the plasma-sprayed hydroxyapatite coatings on Ti-6Al-4V substrate.     

Sensitization of Mice by Inhalation of Antigen After Infection with Bordetella pertussis

Intranasal infection of mice with Bordetella pertussis or injection of pertussis vaccine previous to administration of an albumin aerosol augments sensitivity toward albumin. Sensitization was demonstrated by provocation of anaphylactic reactions following intravenous injection of antigen.     

Effects of Purified Staphylococcal Alpha Toxin on the Ultrastructure of Human and Rabbit Erythrocytes

Previous studies have suggested that the primary site of action of purified staphylococcal alpha toxin is the cell membrane. Scanning and transmission electron microscopy studies were undertaken, therefore, to define toxin-induced alterations in the surface morphology of rabbit and human red blood cells. During the prelytic lag phase, scanning electron microscopy revealed multiple discrete blisters on the surface of rabbit red blood cells; during hemolysis, cellular collapse and ghosts were seen, but most striking was the separation of large fragments of cell membrane from red blood cell surfaces. In contrast, alterations in less sensitive human red blood cells were limited to occasional fingerlike protrusions during the period of accelerated lysis. Transmission electron microscopy substantiated these changes. These studies have provided further evidence that the cell membrane is the primary site of action of staphylococcal alpha toxin.     

Controlled elimination of intracellular H(2)O(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification

The predominant enzymes responsible for elimination of hydrogen peroxide (H(2)O(2)) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H(2)O(2)-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr(90) by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I-IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H(2)O(2) by mechanisms involving the SH3 domains of the kinases and the Pro(293)PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr(231) and Tyr(386). Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.