Prema R. Madyastha

Characterization of interferon gamma receptors on osteoclasts: Effect of interferon gamma on osteoclastic superoxide generation

Osteoclasts are the primary cells responsible for bone resorption. Osteoclast formation and bone resorption activities involve processes tightly controlled by a network of cytokines. The presence of interferon gamma (IFN‐γ) receptors on osteoclasts is a necessary prerequisite for IFN‐γ to directly affect osteoclastic activity. To date, the presence of the IFN‐γ receptor on osteoclasts has not been established. This study provides evidence that osteoclasts express the IFN‐γ receptor. Specific binding of IFN‐γ to the osteoclastic receptor stimulates osteoclastic superoxide generation. The p91 and p47 components of the NADPH oxidase increase after IFN‐γ stimulation and may account for the enhanced superoxide generation. Antisense experiments targeting p91 and p47 subunits abrogate the increased osteoclastic superoxide production stimulated by IFN‐γ. Thus, superoxide generation by osteoclasts is stimulated by activation of a functional IFN‐γ receptor on the osteoclast. J. Cell. Biochem. 84: 645–654, 2002.

An improved method for rapid layering of Ficoll-Hypaque double density gradients suitable for granulocyte separation

A simple technique is described to layer Ficoll-Hypaque double density gradients suitable for rapid isolation of human granulocytes using disposable plastic syringes and 20-gauge needles. The syringes without plungers were kept at 45 degrees angle initially at the top of the test tube, the tip of the needle touching the side of the test tube. While layering the gradients, the syringes were progressively adjusted at 3 different positions which enhanced the smooth flow by forming a continuous stream from the tip of the needle to the top of the lower gradient resulting in excellent interfaces without causing turbulence or inadvertent mixing. This method is useful particularly when unknown sera are screened against granulocytes or lymphocytes from a large number of normal donors.

The use of ficoll-hypaque double density gradients in the separation of avian granulocytes from other cell types for the purpose of cell flow cytometric analysis

Avian peripheral blood and embryonic spleen cells were prepared for cell flow cytometry. The Ortho Spectrum III was the flow cytometer used in these experiments. The major objectives were to identify the location of lymphocytes and granulocytes in the cytogram displayed by flow cytometry, to develop a technique which would allow the collection of granulocytes relatively free of other cell types and to characterize the cell cycle within these cell populations. The cytogram of fresh avian cells developed in the Ortho Spectrum III revealed three characteristic cell clusters. Peripheral blood or embryonic spleen cells were separated on a Ficoll-Hypaque double density gradients into two distinct layers and a pellet. Light microscopic examination revealed the top layer of cells to be primarily lymphocytes while the middle layer of cells was granulocytes. Presentation of the cells from these layers to the Ortho Spectrum III revealed that granulocytes made up Cluster 3 while lymphocytes were included in the other clusters. The Ortho Spectrum III was employed to determine the presence of G1 (pre-DNA synthesis), S (DNA synthesis), and G2/M (post-DNA synthesis and mitosis) phases of cells in Clusters 1 and 2 and Cluster 3. While all the cells from peripheral blood were in G1, the embryonic spleen revealed cells in G1, S and G2/M in both Clusters 1 and 2 and Cluster 3.

Cytophilic immunoglobulin G binding on neutrophils from a child with malignant osteopetrosis who developed fatal acute respiratory distress mimicking transfusion-related acute lung injury

A 16‐month‐old boy, diagnosed at age 3 months with osteopetrosis, was treated since age 6 months with rhlFN‐γ in combination with rhM‐CSF. The child developed acute respiratory distress within 1 hr of a paternal platelet transfusion. Both the child and the father were blood group type O, and platelets were collected the previous day from the father. Chest X‐ray revealed right pulmonary consolidation and a complete “whiteout” on the left. By 24 hr, the lungs had the appearance of adult respiratory distress syndrome (ARDS). Over the course of the next 11 days, the child remained intubated and hypotensive, and died of respiratory insufficiency 11 days later. ARDS was confirmed at autopsy. Pre‐ and posttransfusion patients sera, as well as paternal serum, were tested by granulocyte agglutination and flow cytometry against granulocytes (PMN) from the patient, father, mother, and routine cell‐panel donors and lymphocytes for the presence of neutrophil‐specific and lymphocyte (HLA) antibodies, to rule out classical transfusion‐related acute lung injury (TRALI). Both the patients and the paternal sera were devoid of antibodies, but the patients neutrophils demonstrated strong binding of cytophilic IgG accompanied by extremely low serum IgG and IgG1 levels. Since rhlFN‐γ is known to upregulate Fc gamma receptor type I (FcγRI) with high affinity for IgG1, the binding of cytophilic IgG suggests that the patients neutrophils may have been activated in vivo. The case report of another child with osteopetrosis has also been described. Although the blood specimen was not available for serological studies, this 4½‐year‐old child treated with rhlFN‐γ and rhM‐CSF also died of adult respiratory distress syndrome, with similar clinical presentations.

Incidence of neutrophil antigens on human cord neutrophils

ABSTRACT: Neutrophils isolated from cord blood of healthy newborns (33 blacks and 21 whites) were investigated by EDTA‐microagglutination for their expression of neutrophil specific antigens that have been associated with isoimmune neonatal or autoimmune neutropenia. Equal volumes of various neutrophil anti‐sera (2 μ) and cord neutrophils (3–5 × 106/ml) were mixed in tissue typing microplates under oil and were incubated at room temperature for 6–8 hr, following which the degree of agglutination was noted. Our data revealed that all the currently recognized neutrophil antigens are readily demonstrable by antineutrophil antibodies in cord blood, (NA1, 52–54%; NA2, 81–85%; NB1, 95–96%; NC1, 90%; 9A, 29–30%) suggesting that neutrophil antigens are fully expressed at birth.