Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

PHA-induced human T cell colony formation: enhancing effect of large granular lymphocytes.

The capacity of phytohemagglutinin (PHA)-stimulated human T cells to develop into colonies in agar has been evaluated in the presence or absence of a variety of peripheral blood mononuclear cell subsets. A subpopulation of non-T, non-B cells with receptors for the Fc portion of IgG (i.e. third population cells or TPC) was found to enhance considerably the T cell colony forming capacity. Since TPC have been previously shown to be highly enriched for large granular lymphocytes (LGL, i.e. cells with cytoplasmic azurophilic granules and acid hydrolases), LGL were purified on Percoll density gradients and tested for their T cell colony enhancing capacity. It was shown that LGL were indeed the cells capable of enhancing the T cell colony formation.

Transforming growth factor-beta regulates the splicing pattern of fibronectin messenger RNA precursor.

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions (ED-A, ED-B and IIICS) of the primary transcript of a single gene. Using monoclonal antibodies, we previously demonstrated that transforming growth factor-beta (TGF-beta) preferentially increases the accumulation of the FN isoforms containing the ED-A sequence in cultured normal human fibroblasts [Balza et al., (1988) FEBS Lett. 228, 42-44]. To determine the basis of this effect, we have examined through S1 nuclease analysis, the levels of ED-A- and ED-B-containing mRNAs in cultured normal human skin fibroblasts before and after TGF-beta treatment. These experiments have shown that TGF-beta increases the relative amount of m-RNA for ED-A- and ED-B-containing FN isoforms. These data demonstrate that a growth factor may regulate the splicing pattern of a pre-mRNA.

Analysis of human peripheral blood lymphocytes isolated by counterflow centrifugation-elutriation.

Human peripheral blood mononuclear cells isolated by Ficoll-Hypaque density gradient centrifugation have been fractionated by counterflow centrifugal elutriation (CCE). Six CCE fractions were obtained and subsequently analyzed as for their content of monocytes, T cells, NK cells and B cells. The various cell types were identified through the expression of specific surface membrane determinants or by cytochemical staining for alpha-naphthyl acid esterase (ANAE). Monocytes were elutriated at the highest counterflow rates whereas the majority of B cells were collected at the lowest counterflow rates. T cells as well as NK cells were mostly concentrated in the intermediate fractions. No differences in the elutriation profile of T cells with the helper-inducer or with the cytotoxic-suppressor surface phenotype were observed. However, the percentages of T cells as determined by surface marker expression decreased with increasing counterflow rates, whereas the percentage of ANAE-positive T cells increased. Yet, T cells recovered at the high counterflow rates had ANAE-reactive organelles larger than those of T cells collected at low counterflow rates. These findings suggest that T cells at different maturational stages could be separated by CCE.

Immunofluorescent and ultrastructural analysis of plasma cell degeneration in the chicken Harder's gland.

In this study we describe some aspects of plasma cell degeneration in the chicken Harder's gland. The immunofluorescent patterns of cytoplasmic immunoglobulin (cIg) localization have been studied in relation to the ultrastructure of maturing and degenerating B cells. It appears that Russell body formation through the accumulation of Ig within the cisternae of the rough endoplasmic (RER) does not represent the only mechanism for the formation of cytoplasmic vacuoles. Mitochondrial swelling and disruption or dilation of Golgi cisternae, often preceding alterations of the RER, may be the origin of some vacuoles. It also appears that, in the Harder's gland, degeneration may occur not only in mature plasma cells but also in maturing B cells at a stage when only clusters of polyribosomes are found in the cytoplasm and no RER is yet developed. These observations are relevant to some immunofluorescence and ultrastructural patterns observed in human B-cell pathology.

Structure of 5' region of human tenascin-R gene and characterization of its promoter.

The tenascin-R (TN-R) gene encodes a multidomain extracellular matrix protein belonging to the tenascin family, previously detected only in the central nervous system. In this report, we describe the structure of the 5' region of the human TN-R gene and characterize the activity of its promoter. We cloned two previously unreported nontranslated exons (exons 1 and 2, 539 and 101 bp in length, respectively) separated by a large (> or = 40-kb) intron. The intron between exons 2 and 3 (containing the ATG codon) is 122 kb in length. Tenascin-R transcripts in fetal, adult, and neoplastic human brain contain both exons 1 and 2, as demonstrated by S1 nuclease analysis and reverse transcriptase-polymerase chain reaction. The human TN-R promoter displays relatively unusual features in terms of sequence in that it lacks any TATA box, CAAT box, GC-rich regions, or initiator element. The promoter displays its activity only in cultured cells of neural and glial origin, not in transformed epithelial cells and melanoma cells. All the elements required for the full and cell-specific activity of the promoter are contained in the 57-bp sequence closest to the transcription startpoint.

Morphology, cytochemical features, and membrane phenotype of HLA-DR+ interstitial cells in the human pancreas.

The morphology, histological distribution, surface, and enzymatic phenotype of pancreatic HLA-DR+ cells were studied on seven human pancreata, removed from cadaver donors. Frozen and paraffin-embedded pancreas sections were stained with a battery of monoclonal antibodies by indirect immunofluorescence, immunoperoxidase, and immunophosphatase techniques. Two type of cells were found to express HLA-DR surface molecules: endothelial cells and nonfibroblastic non-B and non-T interstitial elements. The latter cells, which were localized both in the exocrine and endocrine portions of the organ, were distinguished in two main families (macrophagic and dendritic) according to their morphology, surface phenotype, and lysosomal enzymatic activities. The phenotype of cells belonging to macrophagic cell family was the following: Leu M1+, Leu M2+, Leu M3+, OKM1+, and OKT6-. In addition these cells were positive for the expression of lysosomal enzymes such as alpha-naphthyl acetate esterase (ANAE) and acid phosphatase (AP). The "dendritic" cell family comprised, among others, cells that were characterized by the presence of numerous finger-like projections, the absence of Leu M1, Leu M2, Leu M3, OKM1, OKT6 surface antigens, and by the negativity for ANAE and AP. These "dendritic looking cells" (DLC) constituted the most represented DR+ cell type within pancreatic islets. The demonstration of dendritic cells within human islets may justify, in humans also, in vitro procedures of intra-islet dendritic cell removal prior to transplantation, in the attempt of islet rejection prevention.

Autoreactive lymphocytotoxic IgM antibodies in highly sensitized dialysis patients waiting for a kidney transplant: identification and clinical relevance.

The contribution of different families of lymphocytotoxic antibodies in the serologic reactivity of 45 highly sensitized dialysis patients (HSDP) (panel reactivity antibody value-PRA greater than 80%) was assessed by analyzing patients' sera for the presence of auto- and alloreactive IgM and alloreactive IgG antibodies. A total of 220 sera was screened at different incubation temperatures, before and after treatment with the reducing agent dithiothreitol, against a large variety of cell targets by means of complement dependent cytotoxicity (CDC) and antiglobulin augmented (AHG) CDC assays. The results allowed to subdivide the HSDP under study into four groups: Group 1 consisted of 13 untransplanted patients and 14 patients with a prior failed graft whose PRA values did not change following DTT treatment. Alloreactive IgG antibodies alone, with anti-HLA specificity, were present in the sera of this patient group. Group 2 consisted of 3 untransplanted patients whose sera did not contain any autolymphocytotoxic antibody but appeared completely unreactive to panel lymphocytes following DTT treatment, thus confirming the presence of alloreactive IgM only endowed with antiHLA reactivity. Group 3 consisted of 4 untransplanted and 4 patients with a prior failed graft whose sera were found to contain in addition to autoreactive IgM also alloreactive IgG antibodies. Their PRA values declined after DTT treatment on average from 96.2% to 45% and from 95% to 52.5%, respectively. Group 4 consisted of 6 untransplanted patients whose PRA reactivity to both autologous and panel lymphocytes completely disappeared following DTT treatment, thus indicating that their sera contained exclusively autolymphocytotoxic IgM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental study to examine the patency and tissue response of two types of biosynthetic graft used as a replacement for porcine inferior vena cava.

This experimental study has been carried out to evaluate biosynthetic grafts as vascular substitutes. Tubular segments of 35 x 8 mm made of (1) tanned ovine collagen and integral polyester mesh, either of the first (Omniflow I) or second generation (Omniflow II), or (2) polytetrafluoroethylene (e-PTFE), have been sutured in the infrarenal inferior vena cava of pigs, and removed 1 hour, 7, 14, 28, 56 and 112 days after implantation. The patency rate of biosynthetic grafts was higher than that of e-PTFE grafts (p < 0.01). There was no significant difference between the patency of the first generation and second generation collagen grafts. These results indicate that biosynthetic prostheses may be suitable vascular substitutes in low flow and low pressure systems. Improvements in the collagen inner cover structure (Omniflow II vs. Omniflow I), producing greater mechanical endurance, did not enhance long-term patency or the healing patterns of biosynthetic grafts.

[Analysis of the cellular infiltrate and epithelial class I and II molecular expression in edematous type nasal polyps]. / Analisi dell'infiltrato cellulare e della espressione epiteliale di molecole HLA di istocompatibilitá di classe I e II in polipi naso-sinusali di tipo edematoso.

Although in the last few years histopathological, immunohistochemical and immunological studies on nasal polyps have been carried out by several Authors, the etiology of these formations still remains unknown. Nasal polyps have a very characteristic structure and have been classified in three histologic types: edematous, glandular and fibrous. In the present report, 11 nasal polyps of edematous type, representing 61% of total number of collected polyps, were studied employing immunohistochemical methods. All the examined polyps were similar in histology and positivity pattern for HLA molecule expression. The edematous core appeared infiltrated (149 cells/mm.2) mainly by eosinophils (> 90%), whereas the peripheral subepithelial connective tissue contained cellular clusters (416 cells/mm2.) made up of different subsets of hematic cells (30.8% were monocytes-macrophages and 48.6% were lymphocytes largely represented by CD4+ cells). On the contrary, mast cells were quite rare (on the average 1.7 cells/mm2.) and located near T cell clusters. The epithelial positivity for HLA-DR and HLA-A,B,C molecules showed a characteristic discontinuous pattern. In the same patient, controlateral nasal mucosa showed a histological structure very similar to that of polyps. The above data suggest that the presence of polyps is the result of an inflammatory process brought about by a complex array of cellular and humoral components.

[Nasal polyps: comparative immunological study of polyps with different histopathologic types]. / I polipi naso-sinusali: studio immunoistologico comparativo di formazioni con differente tipologia istopatologica.

Cellular Infiltrate as well as class I and II HLA molecule expression, on 22 nasal polyps and on 12 samples of corresponding hypsilateral mucous membrane were studied by means of immuno-histological methods. These nasal polyps were classified according to their histopathological structure. Five polyps, with a fibrous connective core infiltrated by cells of the monocyte-macrophage lineage, were classified mixed. The remaining seventeen polyps were characterized by the presence of central oedematous connective tissue infiltrated almost exclusively by eosinophils and either contained (glandular type) or did not contain (oedematous type) glands. A comparative study of different types of nasal polyps and corresponding hypsilateral nasal mucous membranes was carried out on atopic and non-atopic patients. No correlation between atopic status and polyp presence or polyp typology was found. On the other hand, different polyp types appear to have a structural correlation with the corresponding hypsilateral mucous membrane regarding infiltrate cell type, oedematous or fibrous connective tissue presence and expression of on HLA antigen positivity pattern. The characteristic histological structure of hypsilateral mucous membranes in patients with different types of polyps appeared to be brought about by a multifactorial etiology involving mucosal hyperreactivity. Lastly, both polyps and parapolypal nasal mucous membranes were found to be infiltrated mainly in the peripheral subepithelial connective tissue by lymphocytes (55%) as well as by other leukocyte types. The presence of growth factors capable of enhancing an increase of fibroblasts, endothelial cells, together with focal distrupture on the basal membrane, might well be a general mechanism responsible for polyp sprouting.

Tenascin isoforms: possible targets for diagnosis and therapy of cancer and mechanisms regulating their expression.

Functionally different tenascin (TN) isoforms containing varying numbers of III homology repeats are generated by alternative splicing of a single TN primary transcript. It has recently been reported that the larger TN isoform is, in general, more expressed in neoplastic tissues than in the normal tissues from which the tumor originates. This is due, at least in breast lesions, to the high proliferative activity of stromal elements. In fact, TN splicing is cell-cycle dependent, thus offering a viable system to study the molecular mechanisms that regulate alternative splicing and suggesting that cell-cycle dependent modifications in the splicing pattern of primary transcripts (which very likely are not limited to the TN pre-mRNA) may also be a cell-cycle regulatory mechanism. Furthermore, the very high accumulation of the larger TN isoform in neoplasia allows wider diagnostic and therapeutic monoclonal antibodies specific for the larger TN isoforms be considered for a number of tumors.

[Expression of HLA-DR, DP, DQ antigens in fibrous sino-nasal polyps]. / Espressione di antigeni HLA-DR, DP, DQ, in polipi naso-sinusali di tipo fibroso.

In the last years nasal polyps have been studied by several authors with different methodologies; however, their etiology is still unclear. In this paper we have analyzed in four nasal polyps of fibrous type, the HLA class II (HLA-DR, DP, DQ) molecule expression by means of immunohistochemical techniques (immunoperoxidase and immunophosphatase). A strong inflammatory cell infiltration, a percentage increase of both HLA-DR+ and HLA-DQ+ cells (normal nasal mucous membrane stroma infiltrating cells: DR+ < 40%, DP+ < 2%, DQ+ < 3%; fibrous polyps infiltrating cells: DR+ = 68%, DP+ < 2, DQ+ = 7%) as well as a clear positivity for DR expression of both surface and glandular epithelia were observed in all polyps. Furthermore, in the stalk area of one of the studied polyps DR+DQ+ cells with macrophagic features and having tight. connections with the vessels were observed. The scanty vascularization with the presence of activated mononuclear and mast cells might be responsible for polyp growth by locally producing an anomalous concentration of growth factors.