Methods
Although arthritis occurs in many different forms, loss of articular cartilage and joint inflammation are the predominant pathophysiological processes consistently present in this group of diseases. Depending upon the particular arthritide, these two pathologies may take place together or separately. Evidence is accumulating to suggest that NO is produced locally within human osteoarthritic and rheumatoid...
Macrophages and the various inflammatory mediators they release have been implicated in lung injury induced by a number of different pulmonary toxicants. Exposure of humans or experimental animals to toxic doses of xenobiotics such as ozone, bleomycin, or mineral dusts results in an accumulation of macrophages in the lung. These cells are activated to release increased amounts of proinflammatory and...
A novel family of chemotactic cytokines or chemokines, essential for the directed migration of leukocytes to sites of inflammation, has been identified during the past decade. To obtain microgram amounts of natural chemokines, normal (e.g., freshly isolated leukocytes, connective tissue cell cultures) or malignant cell lines have to be selectively induced with endogenous (cytokines) or exogenous (bacterial,...
Our previous work detailing the antiproliferative effects of nitric oxide (NO) in various assays of lymphocyte functionin vitroled us to examine whether this immunosuppressive effect was also operativein vivo.The efficacy of administration of the NO synthesis inhibitor, aminoguanidine (AG), by various routes was examined. Oral administration as a 1% solution in the drinking water effectively lowered...
Although IL-8 has been reported to be a chemoattractant for T cellsin vivoandin vitro,this has been a controversial issue. By using freshly purified human T cells (>90% CD3 + ), we demonstrated consistent T-cell migration in response to recombinant human IL-8in vitro.However, highly purified T cells, when incubated at 37°C for more than 12 h or cultured overnight in the presence of anti-CD3...
Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin-secreting β cells found in islets of Langerhans. The biochemical mechanisms associated with β-cell destruction have remained elusive. Cytokines, released from T lymphocytes, macrophages, and monocytes during islet insulitis, have been implicated as effector molecules that participate...
Free cytokine and chemokine concentrations in normal human serum are generally below the detection limit. For IL-8, this limit is about 6 pm. In one study, with an ELISA of higher sensitivity, mean serum IL-8 concentration was 2 ± 0.2 pm. Measurement of autoantibody and immune complex in normal human serum suggests a distinction between cytokines and chemokines. Free serum autoantibody to IL-1α, TNF-α,...
Structure/activity relationships underlying the function of monocyte chemoattractant protein-1 (MCP-1) have been probed by site-directed mutagenesis and indicate that the N-terminus of MCP-1 plays a critical role in activating the MCP-1 receptor, especially aspartate-3. However, a monocyte chemoattractant motif analogous to the ELR sequence of neutrophil-active chemokines has not yet been identified...
The role of chemokines in human diseases has been identified primarily by the correlation of increased chemokine protein or mRNA expression during disease development. The identification of increased chemokine expression is only the first step in determining their role in the regulation of inflammatory responses. More recently, several laboratories, including our own, have begun examining the extent...
In this report, we review the evidence for the production of nitric oxide following cytokine activation of the inducible form of nitric oxide synthase by central nervous system glial cells, with particular reference to cells of human origin. The results suggest that cytokine regulation of this enzyme in human glial cells differs significantly from that found in cells of rodent or murine origin, with...
Chemokines activate leukocytes by binding to G protein-coupled receptors on the cell surface. Cloning DNA that codes for chemokine receptors is important because it can lead rapidly to detailed information about their structure, function, expression, regulation, and evolution, information that often cannot be obtained as readily or as precisely by studying the native protein directly. Many cloning...
At present the best way to determine the precise intracellular localization of proteins, in a potentially semiquantitative way, is the combination of ultrathin cryosectioning and immunogold labeling. This paper focuses on the intracellular localization of MHC class II molecules, which are involved in the T helper response to exogenous antigens. Newly synthesized MHC class II heterodimers associate...
Relevant literature relating to immunonegative staining is reviewed and integrated with current research of the author and others. The immunonegative staining procedure has been utilized for the study of epitope localization on immune complexes formed from keyhole limpet hemocyanin type 2 (KLH2) di- and multidecamers, and the 20S and 26S proteasome fromXenopus laevis.The IgG linkage pattern of molecules...
We employed immunoelectron microscopic techniques to localize natural killer cells (NK cells) in human lymph node and tonsil. These tissues were embedded in Lowicryl K4M. Thin sections were first reacted with anti-Leu-7 followed by anti-UCHL. Colloidal gold particles of different sizes were used as a label. NK cells were localized primarily in paracortical T-cell regions. The cells typed with these...
Immunogold–silver staining (IGSS) utilizes a histochemical method called autometallography (AMG) to amplify tiny gold particles to sizes easily visible both in light and electron microscopy. In both applications it is advisable to use the smallest possible gold diameters (1–6 nm) to obtain the highest sensitivity, thus, allowing minute amounts of the target substance to be demonstrated. Gold labels...
Expression of human immunodeficiency virus (HIV) Gag precursor protein (Pr55) by recombinant baculoviruses in insect cells results in the assembly and budding of Pr55 as virus-like particles, or Gag pseudovirions. The ultrastructural morphology, size, and sucrose sedimentation rate of Gag pseudovirions are indistinguishable from immature lentivirus particles produced by HIV-infected human cells. Recombinant...
Phosphoproteins and their associated kinases and phosphatases play a major role in second messenger signal cascades in neurons. Immunocytochemical studies have shown that some of these phosphoproteins and their associated enzymes are homogeneously distributed in brain and function in signal cascades common to all neurons. Other phosphoproteins and their associated enzymes are heterogeneously distributed...
We have demonstrated that unlabeled antibodies or their antigen-reactive fragments, Fab and F(ab′) 2 , can be readily visualized by negative-stain immunoelectron microscopy and can be quite useful as probes for detailed epitope mapping and structural analysis of relatively small macromolecules. In addition, information on segmental flexibility of the target molecules can be deduced as can...
Highly ordered insect flight muscle provides an excellent system for coordinated immunolocalization of sarcomeric proteins at increasing levels of resolution, from fluorescent and gold-tagged secondary antibodies to 3- and 5-nm gold directly coupled to Fab fragments. The penetration of antibody probes of various sizes into native and preserved muscle or tissue sections is compared. Factors affecting...