Effect of allogeneic cell interaction on the primary immune response in vitro. Cell types involved in suppression and stimulation of antibody synthesis

A graft-versus-host (GVH) reaction was induced in F1 hybrid mice by the inoculation of spleen cells from one of the parental strains. One week later the spleen cells from the recipients were cultured during the conditions for obtaining a primary immune response in vitro described by Mishell & Dutton (1967). It was found that the antibody response against the thymus-dependent antigen sheep red cells (SRC), as well as the thymus-independent antigen lipopolysaccharide from Escherichia coli 055:B5 (CPS) was markedly depressed. Spleen cells from mice subjected to a GVH reaction (GVH cells) also inhibited the antibody response of normal cells in vitro. The inhibitory effect of the GVH cells on normal cells was not sensitive to treatment with anti-θ serum, but could be completely abolished by treatment with iron powder, which removes adherent cells.

By culturing cells of two different mouse strains together in vitro it was possible to obtain stimulation or inhibition of the antibody response depending on the total cell number per dish.

The relation of these results to the phenomenon of antigenic competition is discussed. It is suggested that antigenic competition is caused by a non-specific inhibition of cell proliferation, possibly mediated by a locally acting factor. Both thymus-derived and bone marrow-derived cell lymphocytes are affected by the phenomenon. The cells initially responsible for the inhibition seem to be antigen-activated thymus-derived cells (T-cells), which by secondarily activated cells such as macrophages inhibit other cells. A GVH reaction, which generally leads to antigenic competition may, when less pronounced, cause stimulation of the antibody response.

     

Maintenance of allogeneic cell recognition in allograft tolerant mice

The organ distributions of ⁵¹Cr-labelled lymph node cells following transfer to syngeneic, allogeneic and specifically tolerant allogeneic recipients were compared. Neonatal induction of transplantation tolerance in several donor—recipient combinations involving different H-2 haplotypes had no effect on the reduction of homing into lymph nodes and the values of the homing did not differ from those of the untreated allogeneic recipients. In both cases, they amounted to 45–55% of the syngeneic controls. Even those neonatally treated animals, which had not been rendered tolerant at birth and rejected test skin grafts, were not capable of removing the transferred allogeneic cells by a typical immune elimination. The results indicate that animals recognize allogeneic cells, irrespective of whether or not they reject test skin grafts.     

Uroplakins, specific membrane proteins of urothelial umbrella cells, as histological markers of metastatic transitional cell carcinomas.

Uroplakins (UPs) Ia, Ib, II, and III, transmembrane proteins constituting the asymmetrical unit membrane of urothelial umbrella cells, are the first specific urothelial differentiation markers described. We investigated the presence and localization patterns of UPs in various human carcinomas by applying immunohistochemistry (avidin-biotin-peroxidase complex method), using rabbit antibodies against UPs II and III, to paraffin sections. Positive reactions for UP III (sometimes very focal) were noted in 14 of the 16 papillary noninvasive transitional cell carcinomas (TCCs) (88%), 29 of the 55 invasive TCCs (53%), and 23 of the 35 TCC metastases (66%). Different localization patterns of UPs could be distinguished, including superficial membrane staining like that found in normal umbrella cells (in papillary carcinoma), luminal (microluminal) membrane staining (in papillary and invasive carcinoma), and, against expectations, peripheral membrane staining (in invasive carcinoma). Non-TCC carcinomas of various origins (n = 177) were consistently negative for UPs. The presence of UPs in metastatic TCCs represents a prime example of even advanced tumor progression being compatible with the (focal) expression of highly specialized differentiation repertoires. Although of only medium-grade sensitivity, UPs do seem to be highly specific urothelial lineage markers, thus operating up interesting histodiagnostic possibilities in cases of carcinoma metastases of uncertain origin.     

Lymphangiogenesis and expression of specific molecules as lymphatic endothelial cell markers

In recent years, several functional molecules specifically expressed and localized in lymphatic endothelial cells, such as 5'-nucleotidase, lymphatic vessel endothelial receptor-1, vascular endothelial growth factor receptor-3, podoplanin and Prox-1, have been identified. The discovery of the lymphatic endothelial cell markers facilitated detailed analysis of the nature and structural organization of the lymphatic vessels and their growth (lymphangiogenesis). As a result, over the past few years, advances have been made in understanding the cellular and molecular aspects of physiological lymphangiogenesis and tumor-induced lymphangiogenesis. The biology of lymphangiogenesis, particularly the mechanism of its regulation, is very important in understanding the formation of the lymphatic system as a biological regulation system transporting tissue fluid and wandering cells, including lymphocytes, and disease involving lymphangiogenesis. The understanding of the molecular mechanism of lymphangiogenesis and the elucidation of the development of normal and pathological tissues are expected to lead to the development of therapy for intractable diseases, such as malignant tumors and lymphedema.     

Identification of CD14 residues involved in specific lipopolysaccharide recognition.

CD14 is a key molecule responsible for the innate host inflammatory response to microbial infection. It is able to bind a wide variety of microbial ligands and facilitate the activation of both myeloid and nonmyeloid cells. However, its specific contribution to the innate recognition of bacteria is not known. Presently there is no information on the contribution of individual CD14 residues to Escherichia coli lipopolysaccharide (LPS) binding or on the molecular basis of the interaction between CD14 and LPS from other bacteria. LPS obtained from Porphyromonas gingivalis, a bacterium associated with chronic inflammatory disease, binds CD14 and activates myeloid cells but does not facilitate the activation of nonmyeloid cells. The transfer and binding of these two LPS species to soluble CD14 recombinant globulin proteins with single point mutations was examined. Functional activity of the mutant proteins was monitored by E-selectin expression on human umbilical cord endothelial cells. The analysis identified a charge reversal mutation in a single residue, E47, that demonstrated selective binding to E. coli LPS but not to P. gingivalis LPS. E-selectin activation assays indicated that proteins with mutations at position E47 maintained their structural integrity. Other mutations, including a charge reversal mutation of residue E58, did not significantly reduce the binding of either LPS ligand or the ability of the molecule to facilitate E-selectin activation. These data demonstrate that CD14 can selectively recognize different LPS ligands.     

The chicken limb deformity gene encodes nuclear proteins expressed in specific cell types during morphogenesis.

The chicken limb deformity (ld) mutation affects morphogenesis of both limbs and kidneys and is one of few murine mutations for which the affected gene has been isolated. Analysis of the chicken homolog reveals evolutionary conservation of large parts of the encoded ld gene products. This is the first study of these proteins, their intracellular localization, and their temporal and spatial distribution during embryogenesis. A major 180-kD protein is expressed in chicken embryos and certain adult tissues. The proteins are localized in the nuclei of different embryonic cell types in a characteristic punctate pattern. In the developing chicken limb bud, they are expressed in the newly differentiated apical ectodermal ridge and the mesenchymal compartment, where an unequal distribution along the anteroposterior and, subsequently, the dorsoventral axes, is observed. During kidney morphogenesis, expression is initially restricted to the epithelial compartment of the pronephros and mesonephros. These results correlate well with the previous analysis of the murine ld phenotype and imply determinative roles for ld gene products during the morphogenesis of limbs and kidneys. Unexpected expression in the notochord, floor plate, and ventral horns suggests an involvement of the ld gene products in establishment of the dorsoventral polarity of the neural tube.     

Historical markers in the development of allogeneic hematopoietic cell transplantation.

Biol Blood Marrow Transplant 1999;5(6):341-6.     

Parvalbumin,somatostatin and cholecystokinin as chemical markers for specific GABAergic interneuron types in the rat frontal cortex

It remains to be clarified how many classes of GABAergic nonpyramidal cells exist in the cortical circuit. We have divided GABA cells in the rat frontal cortex into 3 groups, based on their firing characteristics: fast-spiking (FS) cells, late-spiking (LS) cells, and non-FS cells. Expression of calcium-binding proteins and peptides could be shown in separate groups of GABA cells in layers II/III and V of the frontal cortex: (1) parvalbumin cells, (2) somatostatin cells, (3) calretinin and/or vasoactive intestinal polypeptide (VIP) cells [partially positive for cholecystokinin (CCK)] and (4) large CCK cells (almost negative for VIP/calretinin). Combining the physiological and chemical properties of morphologically diverse nonpyramidal cells allows division into several groups, including FS basket cells containing parvalbumin, non-FS somatostatin Martinotti cells with ascending axonal arbors, and non-FS large basket cells positive for CCK. These subtypes show characteristic spatial distributions of axon collaterals and the innervation tendency of postsynaptic elements. With synchronized activity induced by cortical excitatory or inhibitory circuits, firing patterns were also found to differ. Subtype-selective occurrence of electrical coupling, finding for potassium channel Kv3.1 proteins, and cholinergic and serotonergic modulation supports our tentative classification. To clarify the functional architecture in the frontal cortex, it is important to reveal the connectional characteristics of GABA cell subtypes and determine whether they are similar to those in other cortical regions.     

Solid-phase radioimmunoassay of cell specific chromosomal proteins

A solid-phase radioimmunoassay has been developed for the detection of cell specific chromosomal proteins which require the presence of DNA for their immunoreactivity. The assay involves the binding of chromatin particles sonicated under highly controlled conditions to PVC plates followed by first and second antibody with extensive detergent washing. The sensitivity and specificity of the assay system compares favourably with the complement fixation technique.     

Stress proteins as molecular markers of neurotoxicity

In response to many environmental and pathophysiologic stressful stimuli, cells undergo a stress response characterized by induction of a variety of proteins, including the heat shock protein family. The inducible heat shock protein 70 (hsp70) is believed to participate in an array of cellular activities, including cytoprotection. Normal brain cells have little detectable hsp70 RNA or protein. However, following a stressful condition hsp70 mRNA and protein are induced in different cell types depending on the severity and the nature of the stimulus. The induction of hsp70 protein correlates with the regional and cellular vulnerability to a particular injury as identified by standard histologic methods. The pattern of hsp70 expression differs in response to various neurotoxic stimuli, including hyperthermia, ischemia, seizures, hemorrhage, and N-methyl-D-aspartate receptor antagonist administration. Hsp70 expression is a useful marker of cellular injury and may help to identify previously unrecognized areas of vulnerability in the nervous system after a neurotoxic stimulus. Hsp70 may also play a neuroprotective role in the brain.     

Loss of allogeneic T-cell activating ability and Langerhans cell markers in human epidermal cell cultures

Human Langerhans cells are the only epidermal cells that express the T6 and HLA-DR antigens and are responsible for the in vitro allogeneic T-cell proliferative responses in the mixed skin cell lymphocyte reaction (MSLR). To investigate the presence of Langerhans cells in normal human epidermal cell cultures, epidermal cell suspensions obtained from normal human skin specimens and from the subsequent epidermal cell cultures were analyzed by indirect immunofluorescence for the presence of T6 and HLA-DR determinants. In parallel, MSLRs were conducted with suspensions of cultured epidermal cells as stimulatory cells. These studies present evidence that when human epidermal cells are grown in culture, they loose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR and T6 antigens. The T6 antigens were lost during the first 2 weeks of culture, while HLA-DR determinants were still expressed by a small number of cells and were progressively lost through duration of cultures. The loss of HLA-DR antigens closely paralleled the progressive inability of human epidermal cells in culture to stimulate allogenic T cells in MSLR.     

Cell cycle related proteins as prognostic parameters in radically resected non-small cell lung cancer

BACKGROUND: Experimental evidence suggests that lung cancer development and progression can be linked to an increased proliferation rate. AIMS/METHODS: To evaluate the immunohistochemical expression of seven components of the cell cycle machinery in a series of well characterised non-small cell lung cancer (NSCLC) specimens (n = 105). RESULTS: Multivariate analysis revealed that simultaneous loss of expression of three of these factors--cyclin D1, the cyclin dependent kinase inhibitor p16, and the tumour suppressor retinoblastoma protein Rb2/p130--correlated with survival, confirming the hypothesis that the cyclin D1-p16-retinoblastoma tumour suppressor pathway is inactivated in most lung cancer samples. CONCLUSIONS: These results suggest that loss of control of cell cycle checkpoints is a common occurrence in lung cancer and support the idea that functional cooperation between different cell cycle regulatory proteins constitutes another level of regulation in cell growth control and tumour suppression.     

Glycine-rich cell wall proteins act as specific antigen targets in autoimmune and food allergic disorders

Our objective was to investigate the presence of a B and T cell immune response directed against the glycine-rich cell wall protein (GRP) in patients with different autoimmune disorders and with food allergy. GRP is an ubiquitous food protein that has high homology with cytokeratins and other self proteins [Epstein-Barr virus nuclear antigen-1 (EBNA-I), heterogeneous nuclear ribonucleoprotein, fibrillar collagen] which are common targets in autoimmune disorders. A peptide (GGYGDGGAHGGGYGG) derived from GRP was used to screen human sera in direct and competitive ELISA assay. Anti-GRP-specific IgG were analyzed for their ability to cross-react with autoantigens. The intracellular cytokine profiles of the peptide-specific T cell clones obtained from representative patients have been studied. BALB/c mice were immunized with the peptide coupled to the carrier protein keyhole limpet hemocyanin (KLH). Serum IgG antibodies directed against the GRP peptide were detected in several autoimmune disorders and in food allergic patients, and were able to cross-react with autoantigens including keratin, collagen and EBNA-I. Twenty-five T cell clones showed a specific proliferative response to the GRP peptide and were of the T(h)0 phenotype. Eight of the 10 BALB/c mice immunized with the peptide coupled to KLH developed an autoimmune response. Our data suggest that phylogenetically highly conserved epitopes in plants, viruses and humans may be responsible for an autoimmune response in susceptible individuals. They also indicate that the antigen spreading of a particular sequence among apparently divergent proteins may participate to initiate or amplify an immune response.     

Acute phase proteins as markers of inflammatory lesions

Acute phase proteins are liver-derived serum proteins, the concentration of which alters in response to infection or inflammation. Cytokines such as interleukin-6, interleukin-1 and tumour necrosis factor- stimulate the liver to produce haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), ₁-acid glycoprotein (AGP) and fibrinogen (Fb) but limit the production of negative acute phase proteins such as albumin. There are interspecies variations in the pattern of response of the acute phase proteins but they are valuable as markers of inflammatory lesions providing diagnostic information for veterinary medicine.Advances have been made in the study of acute phase protein in pigs, dogs and cattle. In the pig, it is evident that CRP is a major reactant whereas Hp is a moderate acute phase reactant. The use of CRP for the diagnosis of inflammatory disease in dogs has been confirmed with studies relating the canine serum CRP concentration to haematological determination of inflammation. CRP assay in canine serum has been shown to confirm primary infectious or inflammatory conditions but can also detect secondary conditions where the primary condition is non-inflammatory. In cattle, the use of Hp assay is now established as a major aid to the diagnosis of infectious and inflammatory disease. Additional benefit to the diagnosis of disease can be provided by analysis of a profile of acute phase proteins such as a combined analysis of SAA, Hp and AGP. The circulating concentration of AGP is maintained at an increased level for a more extended period than that of SAA or Hp in chronic inflammatory conditions.A major advance would be made in the analysis of animal acute phase protein if internationally recognised standards of the proteins in each species were to become available.Originally presented at ECCP 95.     

A lymphoid cell surface glycoprotein involved in endothelial cell recognition and lymphocyte homing in man

We describe a 90-kDa lymphocyte surface glycoprotein, recognized by the monoclonal antibody Hermes-1, that is involved in endothelial cell recognition and lymphocyte trafficking in man. This molecule is selectively expressed on normal or transformed lymphoid cells that are able to recognize and bind to high endothelial venules (HEV, specialized vessels that mediate lymphocyte exit from the blood into lymphoid organs); appears to be linked to HEV recognition function since, in fluorescence-activated cell sorting of variants of a cloned cell line, HEV binding ability co-selects with the Hermes-1 antigen; bears the predominant cell surface epitopes recognized by heterologous anti-human lymphocyte antibodies able to interfere with lymphocyte binding to HEV; and is structurally similar to a previously described mouse lymphocyte surface receptor for HEV. These findings demonstrate that the molecule defined by Hermes-1 either functions as a specific lymphocyte surface receptor for HEV, or is both precisely coregulated and physically and/or functionally associated with such receptors. The expression of this putative receptor for HEV on normal human lymphocyte populations parallels, and thus presumably helps determine, their migratory status in vivo. Hermes-1 should be a powerful tool for analyzing the role of endothelial cell recognition in the traffic of normal and neoplastic human lymphocytes.     

New placental and pregnancy proteins as possible markers in oncology

Summary A number of placental and pregnancy proteins has been detected by immunochemical methods in extracts from human term placentae. Several of these proteins already have been isolated and characterized and are now investigated for their usefulness as markers in oncology.Several of these proteins so long tested appear to be useful as markers in monitoring tumour patients, i.e. in the early detection of tumour recurrence or metastases and in the control of therapy. In addition, we hope that a combination of several of these new markers with already established tumour markers will enable to better diagnose tumours. Also will the use of solitary tissue proteins aid in the differential diagnosis of cancers.