KIM-1 (kidney injury molecule-1) is a type I transmembrane glycoprotein expressed on dedifferentiated renal proximal tubule epithelial cells undergoing regeneration after toxic or ischemic injury. The extracellular domain of KIM-1 is composed of an immunoglobulin-like domain topping a long mucin-like domain, a structure that points to a possible role in cell adhesion by homology to several known adhesion proteins. Two splice variants (a and b), of the human KIM-1 having identical extracellular domains, differ in their cytoplasmic domains and tissue distributions. In this study, we report that the KIM-1b transcript is expressed predominantly in adult human kidney. We describe the generation of 10 monoclonal antibodies against the extracellular domain of human KIM-1, the mapping of their binding sites, and their use in identifying various forms of the protein. We show that human KIM-1b is expressed in adult kidney cell lines, and we demonstrate that a soluble form of KIM-1 is shed constitutively into the culture medium of the cell lines expressing endogenous or recombinant KIM-1b by membrane-proximal cleavage. A monoclonal antibody that binds at or close to the proteolytic site can partially block the shedding of KIM-1. Release of soluble KIM-1 is enhanced by activating the cells with phorbol 12-myristate 13-acetate and can be inhibited with two metalloproteinase inhibitors, BB-94 (Batimastat) and GM6001 (Ilomastat), suggesting that the cleavage is mediated by a metalloproteinase. We propose that the shedding of KIM-1 in the kidney undergoing regeneration constitutes an active mechanism allowing dedifferentiated regenerating cells to scatter on denuded patches of the basement membrane and reconstitute a continuous epithelial layer.

The mechanism by which low affinity adhesion molecules function to produce stable cell-cell adhesion is unknown. In solution, the interaction of human CD2 with its ligand CD58 is of low affinity (500 mM ؊1 ) and the interaction of rat CD2 with its ligand CD48 is of still lower affinity (40 mM ؊1 ). At the molecular level, however, the two systems are likely to be topologically identical. Fluorescently labeled glycosylphosphatidylinositol-anchored CD48 and CD58 were prepared and incorporated into supported phospholipid bilayers, in which the ligands were capable of free lateral diffusion. Quantitative fluorescence imaging was used to study the binding of cell surface human and rat CD2 molecules to the fluorescent ligands in contact areas between Jurkat cells and the bilayers. These studies provide two major conclusions. First, CD2/ligand interactions cooperate to align membranes with nanometer precision leading to a physiologically effective two-dimensional affinity. This process does not require the intact cytoplasmic tail of CD2. Second, the degree of membrane alignment that can be achieved by topologically similar receptors deteriorates with decreasing affinity. This suggests an affinity limit for the ability of this mode of cooperativity to achieve stable cell-cell adhesion at approximately 10 mM ؊1 .Many biologically important events are initiated and sustained through low affinity binding interactions in cell-cell and cell-matrix contact areas. Several models suggest that active intracellular mechanisms are engaged to assist these low affinity interactions (1-3). This intracellular regulation can occur through the cytoplasmic domains of the adhesion molecules as in integrins and cadherins (4 -6). Other models hypothesize that the ectodomains of low affinity adhesion molecules can oligomerize to produce stable arrays that enhance the intrinsic affinity of the ectodomain interaction (7-9). Testing of these models has been limited by the absence of quantitative affinity measurements in contact areas. The fluid mosaic membrane can be viewed as a nearly two-dimensional solution in which diffusion is constrained to the plane of the membrane, with a small third dimension arising from the flexible tethering of the membrane anchored adhesion molecules (10). The contact area between two apposing membranes may have additional depth in the third dimension due to fluctuations in the distance between the two membranes. The thesis of this study is that low affinity adhesion interactions can succeed in forming many bonds between cells by organizing the two apposing membranes so that the depth of the third dimension is small and optimal for the relevant adhesion molecule pair. When the third dimension is minimized by this cooperativity, the adhesion molecule concentration is increased and bond formation is strongly favored.In this study, we tested this hypothesis using the human and rat CD2 adhesion systems. CD2 is an adhesion molecule expressed primarily on T lymphocytes in human, rat and other mammalian species. In all sp...

The T-cell surface glycoprotein, CD4 (T4), acts as the cellular receptor for human immunodeficiency virus, type 1 (HIV-1), the first member of the family of viruses that cause acquired immunodeficiency syndrome. HIV recognition of CD4 is probably mediated through the virus envelope glycoprotein (gp120) as shown by co-immunoprecipitation of CD4 and gp120 (ref.5) and by experiments using recombinant gp120 as a binding probe. Here we demonstrate that recombinant soluble CD4(rsT4) purified from the conditioned medium of a stably transfected Chinese hamster ovary cell line is a potent inhibitor of both virus replication and virus-induced cell fusion (syncytium formation). These results suggest that rsT4 is sufficient to bind HIV, and that it represents a potential anti-viral therapy for HIV infection.

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Type I interferons (IFNs) are helical cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system. To achieve a better understanding of the structural basis for the different activities of ␣ and ␤ IFNs, we have determined the crystal structure of glycosylated human IFN-␤ at 2.2-Å resolution by molecular replacement. The molecule adopts a fold similar to that of the previously determined structures of murine IFN-␤ and human IFN-␣ 2b but displays several distinct structural features. Like human IFN-␣ 2b , human IFN-␤ contains a zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-␤ dimers. However, unlike the human IFN-␣ 2b dimer, in which homologous surfaces form the interface, human IFN-␤ dimerizes with contact surfaces from opposite sides of the molecule. The relevance of the structure to the effects of point mutations in IFN-␤ at specific exposed residues is discussed. A potential role of ligand-ligand interactions in the conformational assembly of IFN receptor components is discussed.

The lymphotoxin-B receptor (LTBR) is a tumor necrosis factor receptor family member critical for the development and maintenance of various lymphoid microenvironments. Herein, we show that agonistic anti-LTBR monoclonal antibody (mAb) CBE11 inhibited tumor growth in xenograft models and potentiated tumor responses to chemotherapeutic agents. In a syngeneic colon carcinoma tumor model, treatment of the tumor-bearing mice with an agonistic antibody against murine LTBR caused increased lymphocyte infiltration and necrosis of the tumor. A pattern of differential gene expression predictive of cellular and xenograft response to LTBR activation was identified in a panel of colon carcinoma cell lines and when applied to a panel of clinical colorectal tumor samples indicated 35% likelihood a tumor response to CBE11. Consistent with this estimate, CBE11 decreased tumor size and/or improved long-term animal survival with two of six independent orthotopic xenografts prepared from surgical colorectal carcinoma samples. Targeting of LTBR with agonistic mAbs offers a novel approach to the treatment of colorectal and potentially other types of cancers.

Objective Poor condition at birth may impact on IQ, although its effect on other measures of neurodevelopment is unclear. The authors' aim was to determine whether infants receiving resuscitation after birth have reduced scores in measures of attention, memory and language skills or the need for educational support at school even in the absence of clinical encephalopathy. Methods Three groups of term infants were identifi ed from the Avon longitudinal study of parents and children: infants resuscitated at birth but asymptomatic for encephalopathy (n=612), infants resuscitated who developed symptoms of encephalopathy (n=40) and the reference infants who were not resuscitated and had no further neonatal care (n=8080). Measures of attention, language, memory and the need for educational support were obtained for children between 8 years and 11 years. Test results (standardised to a mean of 100 and SD of 15) were adjusted for clinical and social covariates. Missing covariate data were imputed using chained equations. Results Infants asymptomatic after resuscitation had similar scores to those not requiring resuscitation for all measures while infants who developed encephalopathy had lower working memory (−6.65 (−12.34 to −0.96)), reading accuracy (−7.95 (−13.28 to −2.63)) and comprehension (−9.32 (−14.47 to −4.17) scores and increased risk of receiving educational support (OR 6.24 (1.52 to 26.43)) than infants thought to be well at birth, although there was little evidence for an association after excluding infants who developed cerebral palsy. Conclusions The authors found no evidence that infants who were resuscitated but remained well afterwards differed from those not requiring resuscitation in the aspects of neuropsychological functioning assessed in this study. Infants who developed neonatal encephalopathy had evidence of worse functioning, particularly in language skills and were more likely to receive educational support at school.

SummarySurface lymphotoxin (LT) is a heteromeric complex of LT-c~ and LT-[3 chains that binds to the LT-I3 receptor (LT-I3-R), a member of the tumor necrosis factor (TNF) family of receptors. The biological function of this receptor-ligand system is poorly characterized. Since signaling through other members of this receptor family can induce cell death, e.g., the TNF and Fas receptors, it is important to determine if similar signaling events can be communicated via the LT-13-tL. A soluble form of the surface complex was produced by coexpression of LT-c~ and a converted form of LT-[3 wherein the normally type II LT-[3 membrane protein was changed to a type I secreted form. Recombinant LT-oq/[32 was cytotoxic to the human adenocarcinoma cell lines HT-29, WiDr, MDA-MB-468, and HT-3 when added with the synergizing agent interferon (IFN) y. When immobilized on a plastic surface, anti-LT-[3-tL monoclonal antibodies (mAbs) induced the death of these cells, demonstrating direct signaling via the LT-I3-R. Anti-LT-[3-R mAbs were also identified that inhibited ligand-induced cell death, whereas others were found to potentiate the activity of the ligand when added in solution. The human WiDr adenocarcinoma line forms solid tumors in immunocompromised mice, and treatment with an anti-LT-~-R, antibody combined with human IFN-~/arrested tumor growth. The delineation of a biological signaling event mediated by the LT-I3-R opens a window for further studies on its immunological role, and furthermore, activation of the LT-I3-R may have an application in tumor therapy.T he TNF family ofligands and receptors is a set of regulatory elements in the immune system (t). TNF was discovered as a cytolytic agent circulating in the blood of endotoxin-stimulated animals (2-4). Originally cloned in the expectation that TNF would be a novel antitumor agent, it was later shown that its primary physiologic function lies in initiating the inflammatory cascade underlying the host's immediate defensive response to infection or stress. More complex immunological functions have been described (5,6). Lymphotoxin (LT) 1 e~ (also called TNF-I3) is a similar cytokine secreted by activated lymphocytes (7) and was originally characterized as having the same functions as TNF. Later, activated T and B cells were found to display LT-c~ on their surfaces in an unusual form compIexed with another member of the TNF family called LT-13 in an . A complex with an apparent 1 Abbreviations used in this paper: LT, lymphotoxin; LT-I3-1L, LT-[3 receptor; MTT, 3-(4,5-dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide; TUNEL, terminal deoxynucleotidyl transferase UTP nick-end labeling; VCAM, vascular cell adhesion molecule.LT-c~2/I31 stoichiometry is also present, but only in minor amounts on human lymphocytes. The major LT-cq/f3 z form does not bind to the known TNF receptors, referred to here as TNF-R55 and TNF-R.75, but rather interacts with another receptor in the TNF family called the LT-[3 receptor 14).Currently, the function of the LT system is poorly character...