Manuel Patarroyo

Laminin isoforms in tumor invasion, angiogenesis and metastasis

Laminins are a growing family of alphabetagamma heterotrimeric proteins, commonly found in basement membranes (BMs). These large molecules promote cell adhesion and migration via integrins and other cell-surface receptors. Over 12 laminin isoforms are presently known. The various isoforms have a cell- and tissue-specific expression and are differentially recognized by integrins. Expression of laminin isoforms in tumors usually reflects expression in their normal counterparts. However, during tumor invasion, loss of the BM barrier occurs and a discontinuous pattern of laminin staining is observed. In carcinomas, tumor cells at the invading front strongly express intracellularly the gamma2 chain, a component of laminin-5. Remodeling of the vascular BM is observed during angiogenesis, and penetration of several BMs occurs during tumor dissemination and metastasis. Thus, disregulated cell-laminin interactions are major traits of malignant disorders.

Leukocyte‐Cell Adhesion: A Molecular Process Fundamental in Leukocyte Physiology

Leukocyte-cell adhesion is a form of physical contact characterized by fast (firm) stickiness between the cells. To analyze the biology and molecular basis of this process, an adhesion-specific assay was developed: the phorbol ester-induced aggregation of human lymphocytes. This rapid and antigen-independent intercellular adhesion requires cellular metabolism, an intact cytoskeleton and extracellular divalent cations, and is mediated by preformed cell-surface proteins referred to as CAMs. Phorbol ester also induces aggregation of monocytes and granulocytes, as well as adhesion of T lymphocytes to either B cells or monocytes and of the leukocytes to vascular endothelial cells. By using the adhesion-specific assay and blocking monoclonal antibodies, several CAMs have been identified, namely the Leu-CAM family (CD11a-c/CD18) and ICAM-1 (CD54). The Leu-CAM family is composed of Leu-CAMa (CD11a/CD18), Leu-CAMb (CD11b/CD18) and Leu-CAMc (CD11c/CD18), three glycoprotein heterodimers made of a common beta-chain and distinct alpha-chains. ICAM-1 is an adhesive ligand for Leu-CAMa. Expression and use of the various CAMs is selective in different types of leukocytes. The Leu-CAMs have been purified and partially characterized. CD18, whose gene is on human chromosome 21, contains 5-6 N-linked complex-type oligosaccharides, and CD11 binds Ca++. Another adhesion pathway is mediated by CD2 and CD58. CD2, a glycoprotein selectively expressed by T cells, is a receptor for CD58, a cell-surface adhesive ligand with broad tissue distribution. Antibodies to the latter CAMs do not block the phorbol ester-induced lymphocyte aggregation. Adhesion is involved in a large variety of leukocyte functions. Anti-Leu-CAM antibodies block induction of IL-2 production and lymphocyte proliferation. Lymphocyte-mediated cytotoxicity is also inhibited. Endogenous NK and LAK cells use Leu-CAMs, ICAM-1 and CD2, and sometimes RGD receptors, to bind and kill tumor cells. Endogenous compounds such as H2O2 and LTB4 also induce Leu-CAM-dependent adhesion in monocytoid cells and granulocytes, respectively, and degranulation of the latter cells is enhanced by the adhesion process. Homologous CAMs have been identified in rabbit and mouse. In in vivo studies in the former species, anti-Leu-CAM antibodies block adhesion of leukocytes to vascular endothelium and thereby their migration into extravascular tissues. The antibodies thus inhibit granulocyte accumulation and plasma leakage in inflammatory lesions, and induce lympho- and granulocytosis, indicating that cell-adhesion contributes to the distribution of leukocytes in the body.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a Cell Surface Protein Complex Mediating Phorbol Ester-Induced Adhesion (Binding) among Human Mononuclear Leukocytes

Phorbol esters rapidly induce aggregation of human mononuclear leukocytes in vitro. Previous studies have indicated that cell surface proteins are involved. We report now that the monoclonal antibody 60.3, either as purified IgG or as Fab fragments, to an antigen common to leukocytes completely inhibited the phorbol ester‐induced intercellular adhesion (binding). No inhibition of cell aggregation was observed with monoclonal antibodies to common leukocyte antigen T 200, T‐cell‐associated antigen, monocyte‐granulocyte antigen, brain granulocyle‐T‐lymphocyte antigen, transterrin receptor, mature T‐cell antigens (mol. wt either 67,000 or 19,000/29,000). T helper/inducer cell antigen, sheep erythrocyte receptor, class I or class II antigens, or T cytotoxic/suppressor cell antigen. The antibody 60.3 did not inhibit stimulation of the cells since the characteristic phorbol ester‐induced morphological changes and phorbol ester‐enhanced cap formation of membrane glycoproteins were readily observed. Two major cell surface polypeptides with apparent molecular weights of 90,000 and 160,000 were immunoprecipitated. We conclude that this protein complex, or at least one of its components, mediates adhesion among mononuclear leukocytes.

Decreased expression of interleukin‐1α, interleukin‐1β, and cell adhesion molecules in muscle tissue following corticosteroid treatment in patients with polymyositis and dermatomyositis

OBJECTIVE To study the effects of immunosuppressive therapy, in particular, corticosteroids, on morphologic signs of inflammation and expression of cytokines, adhesion molecules, and class I major histocompatibility complex (MHC) antigen in muscle tissue from patients with polymyositis (PM) and dermatomyositis (DM) and to correlate the molecular changes with changes in muscle function. METHODS Seven patients with PM and 4 patients with DM underwent muscle biopsy before and after 3-6 months of therapy. Ten of the 11 patients were initially treated with prednisolone 30-60 mg/day. The phenotypes of infiltrating inflammatory cells and the expression of interleukin-1alpha (IL-1alpha) and IL-1beta, adhesion molecules, and class I MHC antigen were studied by immunochemistry. Computerized image analysis was used for quantitation of staining. Muscle function was assessed with a muscle function index score. RESULTS Pronounced improvement of muscle function during the treatment period was noted in 8 of the 11 patients. The changes in muscle function coincided with an almost complete disappearance of inflammatory cells, including CD3+ T cells, in the patients with clinical improvement. These patients also exhibited decreased expression of IL-1alpha, IL-1beta, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), leukocyte function-associated antigen 1alpha, and very late activation antigen 4alpha. Of note, there was persistent expression of IL-1alpha, ICAM-1, and VCAM-1 in capillaries and of class I MHC antigens on muscle fibers in several of the patients who, after corticosteroid treatment, still had muscle weakness despite the disappearance of inflammatory infiltrates. CONCLUSION Changes in the muscle expression of key molecules in the inflammatory process, such as IL-1alpha and IL-1beta, ICAM-1 and class I MHC antigens, showed a consistent but not complete concordance with changes in and status of muscle function in patients with myositis who received the current standard treatment for the disease. These data indicate that it is possible to further evaluate various therapies for myositis using molecular analysis of muscle biopsy specimens obtained on repeated occasions. In addition, the data demonstrate a dissociation between muscle function and degree of inflammatory infiltration in the affected muscles and suggest that the functional defects are more related to the expression of molecules such as IL-1alpha in muscle capillaries than to the mere presence of inflammatory cells in the affected muscles.

Identification of a novel adhesion molecule in human leukocytes by monoclonal antibody LB-2

Monoclonal antibody LB‐2 to a surface antigen on human B cells, lymphoblast, monocytes and vascular endothelial cells largely inhibited adhesion among Epstein Barr virus‐immortalized normal B cells (EBV‐B) and concanavalin A‐stimulated blood mononuclear cells (Con A‐BMC) before and after phorbol ester treatment. The antibody inhibited to a lesser extent phorbol ester‐induced aggregation of monocytes, U937 cells and fresh BMC and had virtually no inhibitory effect on the adhesion among enriched T cells and granulocytes. A surface glycoprotein band of 84 kDa was obtained from EBV‐B cells by immunoprecipitation and gel electrophoresis. Immunological and biochemical studies clearly distinguished this molecule from gp90 and associated glycoproteins which also mediate leukocyte adhesion.

Emerging Rules for Subunit-Based, Multiantigenic, Multistage Chemically Synthesized Vaccines

Seventeen million people die of transmittable diseases and 2/3 of the worlds population suffer them annually. Malaria, tuberculosis, AIDS, hepatitis, and reemerging and new diseases are a great threat to humankind. A logical and rational approach for vaccine development is thus desperately needed. Protein chemistry provides the best tools for tackling these problems. The tremendous complexity of microbes, the different pathways they use for invading host cells, and the immune responses they induce can only be resolved by using the minimum subunit-based (chemically produced approximately 20-mer peptides), multiantigenic (most proteins involved in invasion), multistage (different invasion mechanisms) vaccine development approach. The most lethal form of malaria caused by Plasmodium falciparum (killing 3 million and affecting 500 million people worldwide annually) was used as target disease since many of its proteins, its invasion pathways, and its genome have been described recently. A New World primate (the Aotus monkey) is highly susceptibly to human malaria; its immune system molecules are 80-100% identical to those of its human counterpart, making it an excellent model for vaccine development. Chemically synthesized approximately 20-mer peptides, covering all the P. falciparum malaria proteins involved in red blood cell (RBC) invasion were synthesized by the classical t-Boc technology (based on synthetic SPf66 antimalarial vaccine information for identifying targets) and assayed in a highly sensitive, specific, and robust test for detecting receptor-ligand interactions between high-activity binding peptides (HABPs) and RBCs. HABPs were identified, some in which the molecule displays genetic variability (to be discarded due to their tremendous complexity) and elicits a strain-specific immune response and others that are conserved (no amino acid sequence variation). Conserved HABPs were synthesized in a polymeric form by adding cysteines at their N- and C-terminal ends to be used for monkey immunization. They became nonimmunogenic (no antibodies were induced) nonprotection inducers (monkeys were not protected against P. falciparum malaria challenge with a highly infective strain) suggesting a code of immunological silence or nonresponsiveness for these conserved HABPs. A large number of monkey trials involving a considerable number of Aotus monkeys were performed to break this code of immunological silence by replacing critical residues (determined by glycine peptide analogue scanning) to find that the following amino acid changes had to be made to render them antibody and protection inducing: F<-->R; W<-->Y; L<-->H; I<-->N; M<-->K; P<-->D; Q<-->E; C<-->T. The three-dimensional (3D) structure of >100 of these native modified HABPs (determined by (1)H NMR) revealed that the following structural changes had all to be achieved to allow a better fit into the major histocompatibility complex class II (MHC II)-peptide-TCR complex to properly activate the immune system: alpha-helix shortening, modifying their beta-turn, adopting segmental alpha-helix configuration, changing residue orientation, and increasing the distance of those residues fitting into the MHC II molecules from antigen-presenting cells. More than 100 such highly immunogenic, protection-inducing (against P. falciparum malaria) modified HABPs have been identified to date with this methodology, showing that it could lead to developing a highly effective subunit-based, multiantigenic, multistage synthetic vaccine against diseases scourging humankind, malaria being one of them.

T-Cell Reactivity against Streptococcal Antigens in the Periphery Mirrors Reactivity of Heart-Infiltrating T Lymphocytes in Rheumatic Heart Disease Patients

ABSTRACT T-cell molecular mimicry between streptococcal and heart proteins has been proposed as the triggering factor leading to autoimmunity in rheumatic heart disease (RHD). We searched for immunodominant T-cell M5 epitopes among RHD patients with defined clinical outcomes and compared the T-cell reactivities of peripheral blood and intralesional T cells from patients with severe RHD. The role of HLA class II molecules in the presentation of M5 peptides was also evaluated. We studied the T-cell reactivity against M5 peptides and heart proteins on peripheral blood mononuclear cells (PBMC) from 74 RHD patients grouped according to the severity of disease, along with intralesional and peripheral T-cell clones from RHD patients. Peptides encompassing residues 1 to 25, 81 to 103, 125 to 139, and 163 to 177 were more frequently recognized by PBMC from RHD patients than by those from controls. The M5 peptide encompassing residues 81 to 96 [M5(81–96) peptide] was most frequently recognized by PBMC from HLA-DR7+DR53+ patients with severe RHD, and 46.9% (15 of 32) and 43% (3 of 7) of heart-infiltrating and PBMC-derived peptide-reactive T-cell clones, respectively, recognized the M5(81–103) region. Heart proteins were recognized more frequently by PBMC from patients with severe RHD than by those from patients with mild RHD. The similar pattern of T-cell reactivity found with both peripheral blood and heart-infiltrating T cells is consistent with the migration of M-protein-sensitized T cells to the heart tissue. Conversely, the presence of heart-reactive T cells in the PBMC of patients with severe RHD also suggests a spillover of sensitized T cells from the heart lesion.

Localization of Laminin α4-Chain in Developing and Adult Human Tissues

Recent studies suggest important functions for laminin-8 (Ln-8; α4β1γ1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln α4-chain. Immunoreactivity for the Ln α4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln α4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln α4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln α4- and Ln α2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln α4-chain as Mr 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln α4-chain was found as Mr 180,000 protein.

Intimate Molecular Interactions of P. falciparum Merozoite Proteins Involved in Invasion of Red Blood Cells and Their Implications for Vaccine Design

3. Merozoite Surface Protein (MSP) Family 3662 3.1. Merozoite Surface Protein-1 (MSP-1) 3664 3.2. Merozoite Surface Protein-2 (MSP-2) 3675 3.3. Merozoite Surface Protein-3 (MSP-3) 3677 3.4. Merozoite Surface Protein-4 (MSP-4) 3677 3.5. Merozoite Surface Protein-5 (MSP-5) 3678 3.6. Merozoite Surface Protein-6 (MSP-6) 3678 3.7. Merozoite Surface Protein-7 (MSP-7) 3678 3.8. Merozoite Surface Protein-8 (MSP-8) 3679 3.9. Merozoite Surface Protein-9 (MSP-9) or Acid Basic Repeat Antigen (ABRA) 3679

Species-specific identification of Mycobacterium bovis by PCR

The Random Amplified Polymorphic DNA (RAPD) technique was used in the identification of a species-specific fragment of Mycobacterium bovis. A fragment of approximately 500 bp was amplified from the genome of 15 different M. bovis strains, including M. bovis BCG Pasteur, but was shown to be absent in 26 different mycobacteria and 20 different clinical isolates of Mycobacterium tuberculosis. When the fragment was used as a probe in a Southern blot analysis, several radioactive bands common to M. tuberculosis and M. bovis were observed. However, this fragment hybridized specifically to a 2900 bp EcoRI fragment in the M. bovis genome, but failed to hybridize in either M. tuberculosis or M. avium chromosomal DNA. Based on a partial nucleotide sequence of the 500 bp fragment, two oligonucleotide primers were designed and a PCR assay was developed. Using purified mycobacterial DNA samples, only M. bovis and M. bovis BCG rendered a unique amplification band. This PCR assay is able to detect down to 10 fg purified M. bovis DNA, which corresponds roughly to two bacilli. The assay is also useful for identifying the bacilli directly from uncultured biological samples, such as milk.