Immunohistochemical characterization of human cutaneous mast cells in urticaria pigmentosa (cutaneous mastocytosis)

To clarify the origin and function of human cutaneous mast cells (CMCs), immunohistochemical characterization was done in 19 cases of urticaria pigmentosa (cutaneous mastocytosis) using 9 antibodies (anti-leukocyte common antigen, MX-PanB, anti-lysozyme, anti- alpha 1-antitrypsin, anti- alpha 1-antichymotrypsin, anti-vimentin, anti-neuron-specific enolase, anti-factor VIII-related antigen, and anti-ACTH). CMCs showed positive reactions with anti-alpha 1-antichymotrypsin and anti-vimentin in almost all of the specimens. In more than half of the specimens, CMCs were stained positively with anti-alpha 1-antitrypsin, MX-PanB, and anti-factor VIII-related antigen. Anti-leukocyte common antigen and anti-ACTH also showed positive reactions in some specimens. These results confirm the existence of vimentin filaments in CMCs and suggest a functional role of CMCs in hemostasis via factor VIII. Furthermore, immunohistochemical similarity between CMCs and granulocyte/macrophage-group cells is also suggested.     

Immunohistochemical Investigation of Vimentin, Neuron-specific Enolase, α1-antichymotrypsin and α1-antitrypsin in Adenoid Cystic Carcinoma of the Salivary Gland

Fifty-four adenoid cystic carcinomas (ACC) arising in major and minor salivary glands as well as in normal salivary glands were studied by immunohistochemistry for the presence of vimentin, neuron specific enolase (NSE), α₁-antichymotrypsin (α₁-ACT) and α₁-_- antitrypsin (α₁-_- AT). Five patterns of histological differentiation were found in ACC, and for the cellular components of each, it was possible to establish a special immunohistochemical profile. In ACC, vimentin-positive cells were observed in the outer tubular, cyst-lining and small angular cells. NSE was positive in the myoepithelial cells of normal salivary gland. Neoplastic cells of ACC showed NSE positivity mainly in the small angular cells and partly in the duct luminal cells. α₁-ACT was localized in the intercalated duct cells and serous acinar cells of normal salivary gland, and in the duct luminal cells of ACC. α₁-AT could not be detected in any of the epithelial cells of normal salivary gland. In ACC, eosinophilic hyaline material in the cribriform spaces was positive for α₁-AT, but no positivity was demonstrated in tumor cells. The present study showed that there are at least two populations of tumor cells in ACC: duct luminal cells that express α₁-ACT, thus indicating their ductal character, and small angular cells that express vimentin, characteristic of non-luminal cells. Moreover, our results indicate that α₁-AT is a useful marker of basement membrane-like material.     

α1-Microglobulin Glycopeptides Inhibit Antigen-Specific Stimulation of Human Peripheral Blood Leucocytes

Glycopeptides were prepared from proteolytically digested human and guinea pig α₁ microglobulin (α₁-m) by gel chromatography on Sephadex G-100 and affinity chromatography on concanavalin A-Sepharose. Amino acid analysis showed that the average glycopeptide was a tripeptide containing the amino acids aspartic acid/asparagine, isoleucine, and serine and/or threonine. It has earlier been shown that human α₁-m carries two or three N-glycosidically linked oligosaccharides of the high-mannose type. These preparations of glycopeptides inhibited the proliferative response of peripheral human blood leucocytes in the antigen purified protein derivative. The dose-response relationship of the inhibitions showed a greater specific activity of the glycopeptides than of whole α₁-m. Mild alkali treatment of human α₁-m did not affect its specific inhibitory activity, suggesting that the peptide backbone is of little importance for the immunosuppressive effect of α₁-m. No difference in inhibitory activity was seen between human and guinea pig native α₁-m or α₁-m glycopeptides. Human asialo α₁-m exerted a suppressive effect on the antigen-specific leucocyte proliferation similar to that of native α₁-m.     

Expression of α5 (CD49e) and α6 (CD49f) Integrin Subunits on T Cells in the Circulation and the Lamina Propria of Normal and Inflammatory Bowel Disease Colonic Mucosa

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of α₅ and α₆ integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer α₅ and α₆-positive cells than in the peripheral blood. In addition, there were significantly fewer α₆ integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of α₅ and α₆ integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (α₅β₁) and VLA-6 (α₆β₁) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.     

Interaction and localization of Necl-5 and PDGF receptor beta at the leading edges of moving NIH3T3 cells: Implications for directional cell movement

It was previously shown that platelet-derived growth factor (PDGF) receptor physically and functionally interacts with integrin α_vβ₃, effectively inducing cell movement. We previously showed that Necl-5, originally identified as a poliovirus receptor, interacts with integrin α_vβ₃ and enhances its clustering and the formation of focal complexes at the leading edges of moving cells, resulting in an enhancement of cell movement. We showed here that Necl-5 additionally interacts with PDGF receptor in NIH3T3 cells and regulates the interaction between PDGF receptor and integrin α_vβ₃, effectively inducing directional cell movement. PDGF receptor co-localized with Necl-5 and integrin α_vβ₃ at peripheral ruffles over lamellipodia, which were formed at the leading edges of moving cells in response to PDGF, but not at the focal complexes under these ruffles, whereas Necl-5 and integrin α_vβ₃ co-localized at these focal complexes. The clustering of these three molecules at peripheral ruffles required the activation of integrin α_vβ₃ by vitronectin and the PDGF-induced activation of the small G protein Rac and subsequent re-organization of the actin cytoskeleton. These results indicate a key role of Necl-5 in directional cell movement by physically and functionally interacting with both integrin α_vβ₃ and PDGF receptor.     

Immunosuppressive Properties of α1-Microglobulin

α₁-Microglobulin (α₁m), a serum glycoprotein (26,000 d). was found to impede the proliferative response of human lymphocytes to purified protein derivative (PPD) and tetanus toxoid. The data suggest that, α₁m operates through an unstable suppressor mechanism, which no longer can function after 24 h of preculturing. This effect of α₁m on antigen stimulation did not seem to be due to binding of α₁m to PPD or cells, to altered kinetics of the PPD response, or to non-specific cytotoxicity. In contrast, PPD-induced leucocyte migration inhibition was not reversed by α₁m. α₁m did not cause significant inhibition in experiments in which lymphocytes were stimulated by the mitogens phytohaemagglutinin or concanavalin A. Finally, α₁m had its own leucocyte migration inhibitory effect.     

Expression of Integrin αvβ3 in Gliomas Correlates with Tumor Grade and Is not Restricted to Tumor Vasculature

In malignant gliomas, the integrin adhesion receptors seem to play a key role for invasive growth and angiogenesis. However, there is still a controversy about the expression and the distribution of α_vβ₃ integrin caused by malignancy. The aim of our study was to assess the extent and pattern of α_vβ₃ integrin expression within primary glioblastomas (GBMs) compared with low-grade gliomas (LGGs). Tumor samples were immunostained for the detection of α_vβ₃ integrin and quantified by an imaging software. The expression of α_vβ₃ was found to be significantly higher in GBMs than in LGGs, whereby focal strong reactivity was restricted to GBMs only. Subsequent analysis revealed that not only endothelial cells but also, to a large extent, glial tumor cells contribute to the overall amount of α_vβ₃ integrin in the tumors. To further analyze the integrin subunits, Western blots from histologic sections were performed, which demonstrated a significant difference in the expression of the β₃ integrin subunit between GBMs and LGGs. The presented data lead to new insights in the pattern of α_vβ₃ integrin in gliomas and are of relevance for the inhibition of α_vβ₃ integrin with specific RGD peptides and interfering drugs to reduce angiogenesis and tumor growth.     

Characterization of Monoclonal Antibodies to the αIIbβ3 Integrin on Bovine Platelets

A set of monoclonal antibodies (MoAbs) to leucocyte antigens is an essential tool to identify different cell types and functional membrane molecules involved in immune responses. Since no MoAbs existed to bovine integrins, except against the β₂ subfamily, we generated MoAbs to β₃ integrin after the immunization of mice with bovine platelets. Two MoAbs, IL-A164 (IgG_2a) and IL-A166 (IgG₁), were selected that reacted specifically with bovine platelets and detected the same membrane molecule. The antigen was a heterodimer of two polypeptide chains of 122 kDa and 95 kDa as resolved by SDS-PAGE under reducing conditions. Although the Mr of the smaller subunit is identical to that of β₂ integrin, pre-absorption with an antibody to β₂ (or CD18) did not remove the bovine antigen. Comparing the molecular masses of the two subunits in reduced and non-reduced forms showed a pattern that was similar to that of human GPIIb/IIIa (also called α_IIbβ₃ or CD41a). Reduction of the bovine molecule increased the apparent Mr of the light chain from 76 kDa to 95 kDa, while the heavy subunit changed from 136 kDa to 122 kDa. As with human GPIIb, the decrease in Mr of the α-subunit is probably a result of a small disulphide-linked polypeptide, although no additional evidence for this was detected for the bovine integrin. Sequencing of the N-terminal amino acids of both bovine polypeptides showed identity of the bovine integrin with human GPIIb/IIIa.     

Primary gastric Ki-1 positive anaplastic large cell lymphoma: A report of two cases

Two cases with primary gastric Ki-1 positive anaplastic large cell lymphoma are presented. Morphologic features of both cases involved pleomorphism of the neoplastic cells, fibrosis and lymphatic infiltration. The neoplastic cells in both cases were positive for BerH₂ (CD30), LCA(CD45), lysozyme and alpha-1-antitrypsin (α₁-AT). In additional case, the neoplastic cells were additionally positive for MAC387 and (α₁,-antichymotrypsin (α,-ACT). The neoplastic cells in these cases were negative for L26(CD20), UCHL-1 (CD45RO), DAKO CD3 and epithelial membrane antigen (EMA). According to the results of the phenotypic studies, the authors consider that the neoplastic cells have some of the features of histiocytes.
Both patients at 2 and 8 years after surgery without chemotherapy are disease free. This lymphoma is well known to be frequently misdiagnosed as undifferentiated carcinoma. Although rare in occurrence, recognition of this primary lymphoma in the stomach has a significant clinical implication, as the authors consider that its prognosis might be better than undifferentiated carcinoma of the stomach.     

The HLA-B73 antigen has a most unusual structure that defines a second lineage of HLA-B alleles

Abstract: The nucleotide sequence of cDNA encoding the HLA-B73 antigen was determined; it is unusually divergent, differing from other HLA-B alleles by 44–77 nucleotide substitutions. Features that distinguish the B*7301 heavy chain from other HLA-B heavy chains include multiple substitutions in the α₃ domain and a duplication-deletion within the transmembrane region that increases the length of B*7301 compared to other HLA-B heavy chains. The duplication-deletion is shared with subsets of B alleles from the homologous gorilla (Gogo-B) and chimpanzee (Patr-B) loci. Other unusual features of B*7301 are individually shared with certain alleles of the HLA-A, HLA-C, HLA-F, Gogo-B and Patr-B loci. The B*7301 molecules has sequence elements in common with members of the B7 crossreacting group in the α₁ domain and is shown to possess the ME1 epitope, which is held in common with the B7, B22, B27, B42 and B67 antigens. B*7301 has a unique cysteine at position 270 of the α₃ domain which appears accessible but probably does not form disulphide-bonded B*7301 dimers in cell membranes. B*7301 represents a newly discovered but ancient lineage of HLA-B alleles that appears poorly represented in the modern human population.     

Interactions between Ricinus Agglutinin and Human Plasma Proteins

Ricinus agglutinin purified to homogeneity precipitates certain serum glyco-proteins containing terminal non-reducing galactose residues. Immunoelec-trophoresis of human sera in agarose gel with ricinus agglutinin in the antibody trough gave 2 fusing precipitin lines due to reaction with IgM and haptoglobin. All of 50 monoclonal IgM proteins precipitated with ricinus agglutinin, and the reactive sites were localized to the Fc μ fragment. By affinity chromatography on Sepharose columns containing insolubilized ricinus agglutinin prealbumin, albumin, α₁-lipoprotein, α₁-antitrypsin, α₁-acid glycoprotein, α₁-antichymotrypsin, α₂HS glycoprotein, Ge-globulin, caerulo-plasmin, β-lipoprotein, β₁ C-globulin, and transferrin were not retained, whereas α₂-macroglobulin, haptoglobin, IgM, IgA, and IgG were retained and could be eluted with lactose. Fused rocket immunoelectrophoresis revealed varying degrees of microheterogeneity among the latter proteins; almost all of IgM but only a small fraction of polyclonal IgG was retained on the ricinus column.     

Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human α1β glycine receptors

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR β subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the β subunit was examined using site-directed mutagenesis and expression of recombinant (α₁β^Y413F ) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents ( I _Gly) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I _Gly in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC₅₀= 143 μ m ; cSrc EC₅₀= 74 μ m ). Exogenous cSrc, applied intracellularly, upregulated heteromeric α₁β receptors but not homomeric α₁ receptors. Substitution mutation of the tyrosine to phenylalanine at position β-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type α₁β but not α₁β^Y413F receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the β subunit.     

Rapid Report

Sympathetic vasoconstriction is blunted in the vascular beds of contracting skeletal muscles. We sought to determine whether this blunted vasoconstriction is specific for post-junctional α₁- or α₂-adrenergic receptors. We measured forearm blood flow (Doppler ultrasound) and calculated the vascular conductance (FVC) responses to brachial artery infusions of tyramine (which evokes endogenous noradrenaline release), phenylephrine (an α₁ agonist) and clonidine (an α₂ agonist) in 10 healthy men during rhythmic handgrip exercise (10-15 % of maximum) and during a control non-exercise vasodilator condition (intra-arterial adenosine). Steady-state FVC during exercise and adenosine was similar in all trials (range: 243-272 and 234-263 ml min⁻¹ (100 mmHg)⁻¹, respectively; P > 0.5). During exercise the percentage reductions in FVC in response to tyramine (−24 ± 7 vs. −55 ± 6 %), phenylephrine (−12 ± 8 vs. −37 ± 8 %) and clonidine (−17 ± 6 vs. −49 ± 4 %) were significantly less compared with adenosine (all P < 0.05). The magnitude of the blunted vasoconstrictor responses was similar for both receptor subtypes. These findings are in contrast to those from studies in animals demonstrating that α₂-adrenergic receptor-mediated vasoconstrictor responses are much more sensitive to contraction-induced inhibition than α₁-mediated responses. We conclude that vasoconstrictor responses mediated via both post-junctional α₁- and α₂-adrenergic receptors are blunted in contracting human skeletal muscles.     

A Highly Conserved Phenylalanine in the α, β-T Cell Receptor (TCR) Constant Region Determines the Integrity of TCR/CD3 Complexes

In the present study, we have investigated the importance of a phenylalanine (phe¹⁹⁵) in the Tcr-Cα region on Tcr-α, β/CD3 membrane expression. An exchange of phe¹⁹⁵ with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or valine prohibit completely Tcr/CD3 membrane expression. This seems to be due to a lack of interaction between mutated Tcr-α, β/CD3-γɛ, δɛ complexes and ζ₂ homodimers. The Tcr-Cα region around phe¹⁹⁵ seems together with the same region in the Tcr-Cβ region to constitute an interaction site for ζ₂ homodimers. The presence of phe¹⁹⁵ on both Tcr-Cα and Tcr-Cβ causes high avidity interaction with ζ₂ homodimers, whereas his¹⁹⁵ in both Tcr-Cγ and Tcr-Cδ results in an apparently lower avidity interaction with ζ₂ homodimers. It is suggested that the phe¹⁹⁵ region (on β-strand F) and eventually adjacent aromatic amino acid residues on β-strand B region may play an important role in Tcr-α, β/CD3 membrane expression, in Tcr-α, β/CD3 competition with Tcr-γ, δ/CD3 complexes for ζ₂ homodimers and in the control of formation of 'mixed' Tcr heterodimers.     

A VH-Associated Idiotype in Human Anti-Tetanus Antibodies

An anti-idiotypic (α Id) serum was induced against anti-tetanus toxoid (α TET)-reactivc F(ab')₂ fragments of a single donor. After appropriate adsorption the serum was shown to react only with α TET F(ab')₂ fragments and not with F(ab')₂ fragments deplcted of α TET reactivity or anti-diphtheria toxoid (DIP) or anti-purified protein derivative (α PPD) F(ab')₂ fragments of this donor. Inhibition studies with soluble TET indicated that the anti-idiotypic serum partly reacts with determinants close to or within the antigcnic binding site. When tested on a small panel of α TET F(ab')₂ fragments derived from unrelated donors, one out of four donors tested showed cross-reactivity. When tested on α TET F(ab')₂ fragments derived from related family members, the donor's father and two sisters were found to cross-react, whereas the mother and two other sisters did not. The observed cross-reactivity was independent of the litres of α TET antibodies in the serum. Furthermore, two-dimensional gel electrophoretic studies carried oui on α TET IgG of Id-positive or Id-negative family members indicated that the expression of idiotypic determinants is linked to a particular heavy chain. Immunofluorescent staining with α Id and cytofluoro-graphic analysis of donor T-lymphoblast proliferation in response to TET showed that the T cells did not express Id determinants found on his α TET antibodies.     

Conformation of the T-Cell Antigen Receptor-β Chain C-Domain Contributes to Vβ3 Epitope Recognition by Monoclonal Antibody KJ25

The clonotypic T-cell antigen receptor (TCR)-β chain contains two extracellular intrachain disulfide bonds. It belongs to the immunoglobulin gene superfamily and is subdivided into variable (V), joining (J), diversity (D) and constant (C) region. Monoclonal antibody (MoAb) KJ25 is believed to recognize an epitope in the V-domain of TCR-β (Vβ₃) chain, but its epitope requirements are unknown. In this study of TCR-αβ chain interactions using chimeric recombinant TCR-β chains, the authors found that partial substitution of the Cβ-domain with that of interleukin-2 receptor α chain (Tac) sequences led to the loss of TCR-Vβ₃ epitope recognition by KJ25. These results suggest that epitope recognition of the TCR-Vβ₃ by KJ25 MoAb is dependent not only on the V-domain, but also on the close contact with the extracellular C-domain which influences the conformation and epitope recognition of the Vβ₃-region. This may not be unique to Vβ₃ and may be a general feature of TCR-β protein folding.     

Mannan-Binding Protein Forms Complexes with α-2-Macroglobulin. A Proposed Model for the Interaction

We report that α-2-macroglobulin (α₂M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The α₂M/pMBP-28 complexes were isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose. The occurrence of α₂M/pMBP-28 complexes was further indicated by crossed immunoelectrophoresis and by use of an anti-α₂M affinity column and chelating Sepharose loaded with Zn²⁺. The eluates from these affinity columns showed α₂M subunits (94 and 180 kDa) and pMBP subunits (28 kDa) in SDS-PAGE, which reacted with antibodies against α₂M and pMBP-28, respectively, in Western blotting. Furthermore, the α₂M/pMBP-28 complexes were demonstrated by electron microscopy, Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted with anti-C1 s antibodies in ELISA, one of about 650–800 kDa, which in addition contained pMBP-28 and anti-α₂M reactive material, the other with an M_r of 100–150 kDa. The latter peak revealed rhomboid molecules (7 × 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing conditions. This band was also seen in eluates from the anti-α₂M and chelating Sepharose columns. Based on these observations and previous findings by other investigators of a serine protease with about 67 kDa subunits which copurifies with human MBP we propose a model for the interaction of pMBP-28 with α₂M.     

Immunoglobulins and Other Serum Proteins in Feces from Infants and Children

IgA in variable amounts was found in extracts of feces from all of 14 infants and children with no apparent diseases involving the gastrointestinal tract or the immune system, whereas IgG and IgM were only found in some. No age variation was evident's even one-month-old infants seemed fully able to secrete intestinal immunoglobulins. Freeze-drying of the feces facilitated the extraction, and the amounts of immunoglobulins were related to the dry weight of fecas. Most of the immunoglobulins were easily dissolved in saline. Of other serum proteins α₁ antitrypsin was constantly present; α₁ antichymotrypsin and α₂ macroglobulin were found in many. Only traces of albumin were sometimes demonstrated.     

Fibronectin Co-Stimulates via the α5β1 Receptor IL-2, IL-4 Production by Splenic, Granuloma Lymphocytes of Schistosoma mansoni Infected Mice

In murine Schistosomiasis mansoni, soluble worm egg antigens (SEA) induce L3T4⁺ T helper cell-mediated chronic granulomatous inflammations around parasite eggs. Within the fully developed granuloma lymphocytes, macrophages, and eosinophils, fibroblasts are embedded in extracellular matrix (ECM) composed of fibronectin, laminin, glycosaminoglycans and collagens. The present study examined in vitro the putative co-stimulatory role of fibronectin (FN) in acute and chronic infection splenic and granuloma lymphocyte responses. Plate-bound FN enhanced the anti-CD3 MoAb stimulated normal and acute or chronic infection splenic lymphoproliferation by 20–32%. The co-stimulatory effect was evident in SEA stimulated acute but not chronic infection spleen cells. Proliferation of stimulated granuloma lymphocytes could not be up-regulated by immobilized FN. Plate-bound FN significantly enhanced IL-2 and IL-4 production by SEA-stimulated acute, but not chronic, infection granuloma lymphocytes. However, FN had no influence on the high level of IL-2, IL-4 production of anti-CD3 MoAb stimulated acute or chronic infection splenic or granuloma lymphocytes. Because in the antigen-stimulated acute infection spleen or granuloma cultures the co-stimulatory effect by FN was abrogated by the tripeptide (RGD) arg-gly asp, and anti α₅β₁ antibody, enhancement is attributed to signalling via the α₅β₁ integrin receptor of lymphocytes.