[28-year old female patient with respiratory insufficiency, elevated liver enzymes, pancytopenia and fever]. / 28-jährige Patientin mit respiratorischer Insuffizienz, erhöhten Leberwerten, Fieber und Panzytopenie.

We report on a 28-year old female patient with fever and severe respiratory insufficiency requiring mechanical ventilation. Cytomegalovirus pneumonia was diagnosed by bronchoalveolar lavage, and antiviral therapy was initiated. However fever persisted and laboratory workup showed pancytopenia and elevated liver enzymes. Additional blood tests demonstrated a markedly elevated ferritin level and high concentrations of inflammatory cytokines. A diagnosis of hemophagocytic lymphohistiocytosis was made and immunosuppressive therapy was started. The patient's condition and laboratory values improved rapidly.

Role of beta3 integrins in melanoma cell adhesion to activated platelets under flow.

Mechanisms mediating tumor cell attachment to the vessel wall under flow conditions are largely unknown. Therefore we analyzed the ability of human melanoma cells to adhere to an immobilized matrix during blood flow and determined the role of platelets in this process. In a parallel plate flow chamber, M21 melanoma cells were suspended in human blood and perfused over a collagen I matrix at a wall shear rate of 50 s-1 (2 dynes/cm2) to simulate venous flow over a thrombogenic surface. Melanoma cell interaction with the matrix or blood cells and platelets was monitored and quantified by fluorescence and confocal laser microscopy. Despite their ability to adhere to collagen I under static conditions, M21 cells failed to attach directly to this matrix during blood flow. However, they associated with adherent thrombi, and this resulted in stable melanoma cell arrest. Inhibition of platelet activation or platelet integrin alphaIIbbeta3 function abolished M21 cell attachment. Melanoma cell interaction with thrombi was specific and required beta3 integrin expression. M21-L cells which lack integrin alphavbeta3 failed to associate with thrombi and to arrest during blood flow. Transfection of these cells with the integrin subunits alphav or alphaIIb resulted in variants expressing alphavbeta3, as in the wild type, or alphaIIbbeta3. Both variants were able to associate with thrombi and to arrest during blood flow. Therefore, beta3 integrin-mediated binding to activated platelets represents an efficient mechanism for melanoma cell arrest under flow, and this may contribute to the role of platelets in hematogenous metastasis.

Further studies on cell adhesion based on Le(x)-Le(x) interaction, with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells, and adhesion of various tumour cells based on Le(x) expression.

We previously proposed specific interaction of Le(x) (Gal beta 1-->4 [Fuc alpha 1-->3]-GlcNAc beta 1-->3Gal) with Le(x) as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggens et al., J. Biol Chem (1989) 264:9476-9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le(x) or other epitopes, and affinity chromatography on Le(x)-columns of multivalent lactofucopentaose III (Le(x) oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le(x)-expressing tumour cells vs their Le(x)-non-expressing variants showed that only Le(x)-expressing cells adhere to Le(x)-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le(x) determinant in F9 cells is not GSL but rather polylactosaminoglycan ('embryoglycan'), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca2+, and reversible dissociation in the absence of Ca2+ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le(x)-Le(x) interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le(x)-Le(x) interaction based on minimum energy conformation with involvement of Ca2+ is presented.

Calcium-binding proteins 33 kDa, 35 kDa, and 65/67 kDa in normal rat and Morris hepatoma tissues. A biochemical and immunohistochemical study.

Polyclonal antibodies were raised against membrane-associated calcium-binding proteins (apparent molecular masses 65000 and 67000 (CBP 65/67) and 33000 and 35000 (CBP 33 and CBP 35)), which were isolated from rat liver and Morris hepatoma. Using immunoblotting, various amounts of CBP 33 and CBP 35 as well as CBP 65/67 were detected in most rat organs. Using alkaline phosphatase and monoclonal-anti-alkaline phosphatase antibodies (APAAP), all the calcium-binding proteins were detected by immunohistochemical techniques in the plasma membranes of many cells, such as vascular endothelial cells, lymphocytes, epididymal principal cells, secretory and excretory duct cells of certain exocrine glands, straight distal tubular cells of the kidney, and in the cytoplasm of muscle cells and fibres as well as nerve cells and chondrocytes, and in connective tissue elements. Immunohistochemical analysis also showed that in polarized epithelial cells, e.g., renal tubular cells, epididymal principal cells or excretory duct cells, these calcium-binding proteins are present exclusively or mostly in the luminal plasma membrane.

Isolation of immunoglobulins and their use in immunoaffinity HPLC.

For the isolation of monoclonal and polyclonal antibodies different high performance liquid chromatography (HPLC) and high performance affinity chromatography (HPAC) methods were investigated. Specially designed "mixed-bed" ion-exchange and hydroxylapatite columns as well as hydrophobic interaction columns were efficiently applied to the isolation of monoclonal antibodies. When these methods are used for the isolation of polyclonal antibodies from antiserum, the sample has to be pre-treated, e.g. by removal of serum albumin. Protein A HPAC is an easy method and quick to handle, especially for the preparative isolation of antibodies. The antibodies that do not bind to protein A, can be purified by protein G HPAC. If this method cannot be used because of the rather extreme elution conditions, hydroxylapatite, ion-exchange or hydrophobic interaction HPLC have to be considered as alternatives. We further concentrate on immunoaffinity HPLC with immobilized antibodies. This method has proved to be very effective for one-step isolation of antigens, even from very complex samples such as plasma membrane extracts. The problem with immunoaffinity HPLC is the quick deterioration of the columns, caused by increasing denaturing of the immobilized antibodies during elution. In order to solve this problem, an indirect method is recommended for analytical immunoaffinity HPLC. For this purpose, the antibodies are bound to a protein A HPAC column. The solution containing the antigens is then applied. After washing, the antigen-antibody complex is eluted from the column.

High-performance concanavalin A affinity chromatography of liver and hepatoma membrane proteins.

Although the separation of water-soluble glycoproteins by high-performance (HP) concanavalin A (ConA) affinity chromatography (AC) is feasible, irregularities may be encountered with hydrophobic glycoproteins. The separation of plasma membrane glycoproteins from liver and Morris hepatoma 7777, used as a model, showed that not only the interaction between the lectin and the oligosaccharide portion of the glycoproteins plays a role in the chromatographic process, but also the hydrophobic interactions between sample and lectin and between sample and support. In this, the characteristics of the support, such as surface hydrophobicity and pore size, play an important part. It was found that a portion of the ConA is not covalently bound to the column, especially when elution is carried out with buffers containing detergents. Moreover, some extremely hydrophobic proteins could only be eluted from the column when high concentrations of detergents [1% (w/v) or higher] were applied. Despite these difficulties, four membrane glycoproteins from the liver with apparent molecular weights of 60, 80, 100 and 110-120 kilodaltons could be highly enriched by ConA HPAC. These proteins were further fractionated according to their strength of binding to the ConA and their different hydrophobic characteristics, using various detergents as eluents.

High-performance liquid affinity chromatography of liver plasma membrane proteins.

Plasma membrane proteins from liver were analysed by concanavalin A affinity and immunoaffinity high-performance liquid chromatography. In the method, four peptide bands with apparent molecular weights of 140,000, 120,000, 80,000 and 60,000 could be isolated. In the second method, with two immobilized monoclonal antibodies, two corresponding antigens--the membrane proteins dipeptidyl-peptidase IV and GP 110--could be highly purified from plasma membrane extract with good yield in only one step.