Immunohistochemical investigations of lymphocytes in the lymphoid organs of cyclophosphamide treated chickens

The localization of lymphocytes in the lymphoid organs of cyclophosphamide (Cy) treated chickens and untreated control chickens was compared immunohistochemically using a variety of monoclonal antibodies (CT3, 2-6, 11-39, TCR1, TCR2, TCR3, L22, 11G, 3E8, B-4D-4, A-13). In the Harderian glands of Cy treated chickens, an an increase of T cells was observed, though T cells were a few in untreated controls. These increased T cells consisted of CD4 positive or CD8 positive cells. Further, these T cells were stained with TCR2 or TCR3 antibody, and a small number of cells were stained with TCR1 antibody. In other lymphoid organs such as the bursa of Fabricius, thymus, spleen and cecal tonsils, B lymphocyctes severely decreased or disappeared in Cy treated chickens, though no significant alteration in T cell distribution was observed.     

Reactivity of rat anti-Thy-1 serum with peripheral mouse T lymphocytes

Carcinoma of the colon in a 39-year-old woman which caused obstruction and inflammation of the appendiceal lumen remained undetected until postappendectomy complications prompted reevaluation and appropriate surgical intervention. Malignant lesions of the colon, especially the right side, are occurring with increasing frequency in young persons, and delay in recognition and definitive treatment is responsible for poor survival. A high index of suspicion in patients with atypical presentation of appendicitis or unusual operative findings should provide an opportunity for early diagnosis of underlying carcinoma.     

Engraftment of human kidney tissue in rat radiation chimera: I. A new model of human kidney allograft rejection

BACKGROUND: We have recently shown that lethally irradiated normal strains of mice and rats, reconstituted with bone marrow from severe combined immune deficiency (SCID) mice, can be engrafted with human peripheral blood mononuclear cells (PBMC). METHODS: The feasibility of transplanting human renal tissue under the kidney capsule of the SCID/Lewis and SCID/nude radiation chimera and the effects of intraperitoneal infusion of allogeneic human PBMC on the human renal implants were investigated by histology, electron microscopy, immunohistochemistry, and fluorescence-activated cell sorter analysis. RESULTS: Sequential evaluation of the human renal implants from 10 days to 2 months after transplantation showed that human parenchymal elements survive in the implants up to 2 months after transplantation. The overall architecture of the transplanted kidney tissue and the normal structure of individual cells in the glomeruli and tubuli were preserved. Infusion of allogeneic human PBMC after kidney implantation resulted in patchy cellular infiltrates, composed mainly of activated human T cells, and led to prompt rejection of the human renal tissue, whereas no signs of inflammation were observed in human renal implants of chimeric rats that did not receive human PBMC. Treatment with OKT3 antibody, anti-human CD25 antibody, or CTLA4Ig fusion protein in vivo ameliorated the rejection process. CONCLUSIONS: Human adult kidney fragments transplanted into SCID-like rats transiently retain competent parenchymal structures. When these grafts are combined with allogeneic human PBMC, acute cellular rejection develops. We suggest that this chimeric model might be useful for the investigation of the effects of experimental manipulation on the kinetics of the inflammatory response during human renal allograft rejection.     

Detection of apoptosis in human and rat ovarian follicles

OBJECTIVE: The purpose of this study was to determine whether cells acquired from individual human preovulatory follicles undergo apoptosis (physiologic cell death) and, if so, to correlate the degree of apoptosis with characteristics of the follicles or the oocytes derived from the follicles. METHODS: We devised a sensitive nonradioactive method for detecting apoptotic DNA fragmentation in small numbers of cells derived from rat atretic follicles and follicular aspirates of patients undergoing assisted reproductive technologies. RESULTS: Using this method, apoptotic DNA was detected in rat atretic follicles, with optimal detection at 10-100 ng. Furthermore, apoptotic DNA was detected in some, but not all individual human follicular aspirates from several patients, and was found in follicles that produced oocytes that fertilized and developed into embryos. CONCLUSION: Apoptosis occurs in cells from human ovarian preovulatory follicles and may be a normal physiologic process of the follicle during luteinization.     

Localization of corticotropin-releasing factor in primary and secondary lymphoid organs of the rat

Cells of the immune system produce a variety of neuropeptides or peptide hormones, either constitutively or upon induction, and possess specific neuropeptide receptors that display ligand-receptor interactions similar to those described in the central nervous system (CNS). These findings suggest that specific subsets of lymphoid cells can produce and respond to peptides previously thought to be principally neural mediators. Recently, corticotropin releasing factor (CRF) mRNA was detected in the rat thymus and spleen, although the cells that synthesize CRF were not identified. We examined the localization of CRF and its mRNA in the rat spleen, thymus, and mesenteric lymph nodes using immunocytochemistry (ICC) and in situ hybridization (ISH), respectively. Immunoreactive CRF was present in cells in the marginal zone and red pulp of the spleen, in connective tissue septa and the subcapsular region of the thymus, and in the medullary cords and sinuses of the mesenteric lymph nodes. Dual ICC/ISH for CRF and its mRNA, respectively, demonstrated CRF mRNA over CRF-immunoreactive cells, suggesting CRF synthesis. Double-label ICC for CRF and markers for specific immunocyte subsets suggest that CRF+ cells in the spleen and thymus are macrophages. CRF+ cells in primary and secondary lymphoid organs reside in compartments that are innervated by sympathetic nerves, and some cells appears to be contacted by noradrenergic sympathetic nerve fibers, suggesting that CRF release may be influenced by the sympathetic nervous system, as it is in the hypothalamo-pituitary-adrenal axis. The presence of CRF in organs of the immune system suggests that this neuropeptide may modulate immune functions after paracrine release.     

Endoglin (CD 105) expression in human lymphoid organs and placenta

Endoglin (CD 105) is a cell surface antigen widely expressed on vascular endothelium, syncytiotrophoblast, some tissue macrophages, certain culture cells (including early leukemic B-lineage) and some endothelial cell lines. Though its relation to the transforming growth factor-beta (TGF-beta) receptor system is well documented, its function and detailed pattern of expression still remain to be clarified. We examined the differential tissue distribution of endoglin in human lymphoid organs and placenta with several anti-CD 105 monoclonal antibodies (mAbs) using an indirect immunoperoxidase technique, and performed semi-quantitative measurements using an image-analyzing system for comparison. Arterial, venous and capillary endothelia in these organs were reactive with anti-CD 105 mAbs at varying intensities. Interestingly, a distinctly stronger staining pattern was observed in the high endothelial venules (HEVs) which may indicate a special role for endoglin in lymphocyte trafficking. Syncytiotrophoblast expressed endoglin strongly on their apical cell membrane. Extravillous trophoblasts at certain locations selectively expressed endoglin on their cell membranes, suggesting a special role for this surface antigen during trophoblast differentiation.     

B and also T lymphocytes migrate via gut lymph to all lymphoid organs and the gut wall, but only IgA+ cells accumulate in the lamina propria of the intestinal mucosa

In pigs the lymphocytes emigrating from the intestinal wall were collected by cannulating the lymphatics, labeled in vitro using a fluorescent dye and retransfused. The injection of 6.6+/-4.2 x 10(8) cells resulted in a labeling index between 1.5% in intestinal lymph, 0.2% in the spleen and lymph nodes, approximately 0.1% in the intestinal lamina propria and 0.003% in intraepithelial lymphocytes. About 25 % of the injected cells were present in the blood and 1 % was recovered in the lymph. T cells were found in similar proportions in the injected and the recovered cells in the organs (70-80%). The proportion of IgA+ cells among the immigrated cells in the intestinal lamina propria ranged from 5 to 8%, which in absolute numbers was up to 60% of the injected IgA+ cells. T and IgM+ cells did not show a higher accumulation in any organ. These experiments in conventional, unrestrained animals revealed that (1) T cells immigrate into the intestinal lamina propria, (2) preferential migration of IgA+ cells from gut lymph to the intestinal lamina propria is obvious under in vivo conditions and (3) the immigrated IgA+ cells represent a very small population which is difficult to detect when analyzed in relative numbers.     

Differential effects of kynurenine and tryptophan treatment on quinolinate immunoreactivity in rat lymphoid and non-lymphoid organs

Quinolinate is a tryptophan metabolite and an intermediary in nicotinamide adenine dinucleotide (NAD+) synthesis in hepatocytes. Kynurenine is an upstream metabolite in the same biochemical pathway. Under normal physiological conditions, kynurenine is thought to be produced primarily in the liver as an NAD+ precursor. However, during immune stimulation or inflammation, numerous extrahepatic tissues convert systemic tryptophan to kynurenine, and its concentration subsequently rises dramatically in blood. The fate and role of extrahepatic kynurenine are uncertain. In order to begin addressing this question, the present study was performed to determine which cell types can produce quinolinate from either systemic tryptophan or kynurenine. By using highly specific antibodies to protein-coupled quinolinate, we found that intraperitoneal injections of tryptophan led to increased quinolinate immunoreactivity primarily in hepatocytes, with moderate increases in tissue macrophages and splenic follicles. In contrast, intraperitoneal injections of kynurenine did not result in any significant increase in hepatocyte quinolinate immunoreactivity, but rather led to dramatic increases in immunoreactivity in tissue macrophages, splenic white pulp, and thymic medulla. These findings suggest that hepatocytes do not make significant use of extracellular kynurenine for quinolinate or NAD+ synthesis, and that, instead, extrahepatic kynurenine is preferentially metabolized by immune cells throughout the body. The possible significance of the preferential metabolism of kynurenine by immune cells during an immune response is discussed.     

Defective granzyme B gene expression and lytic response in T lymphocytes infiltrating human renal cell carcinoma

BACKGROUND: For over 60 yr, researchers and engineers have based investigations and the design of cockpit displays and structures upon the presupposition that during flight the pilot maintains a head alignment coincident with the aircraft's vertical axis (z-axis). Recent simulator studies have verified the existence of a pilot neck reflex which refutes this long-standing assumption. This reflex, named the opto-kinetic cervical reflex (OKCR), occurs during visual flight and is theorized to be an attempt by the pilot to stabilize a retinal image of the horizon to maintain spatial orientation. As a result, during initial banking maneuvers, pilots view a fixed-horizon image and not a moving-horizon. The research objectives were to determine if the OKCR occurs during actual flight of high performance jet aircraft and to model the response. HYPOTHESIS: Pilots of high performance aircraft will exhibit the OKCR. Additionally, the OKCR is dependent on the phase of banking (entering into or exiting from a banked position). METHODS: This was an observational study in which the head positions of nine pilots were recorded during actual F-15 aircraft flight and subsequently analyzed. RESULTS: Objective data indicate the OKCR caused pilots to tilt their heads during aircraft bank (p < 0.0001). Also, the reflex was found to be independent of the bank phase. CONCLUSION: The OKCR was shown to be a strong, natural response and the flight results correlated closely with simulator results. The effect of these results on pilot training, spatial disorientation, physiological injury and safety, and the redesign of displays for aircraft attitude and virtual reality are discussed.     

A defect in beta-galactosidase of B lymphocytes in the osteopetrotic (op/op) mouse

Tweezer-like receptor molecules have proven their potential for molecular recognition on several occasions. We decided to make twofold use of this receptor design: firstly to learn whether simple molecular forceps consisting of two peptide chains linked by a spacer are able to selectively bind to small peptides, and secondly to investigate the importance of structural preorganization for the characteristics of the receptors. We prepared two encoded combinatorial libraries based on this design, featuring two combinatorial tripeptide chains held by different scaffolds: the use of chenodeoxycholic acid as spacer provided a rigid scaffold for the forceps, whereas linking the peptide chains by a pentamethylene chain yielded a very flexible forceps structure. Molecules from the cholic acid library recognize and discriminate various enkephalins with micromolar affinities. Molecules from the flexible library show distinct interactions with the enkephalins as well, but the specificity and affinity are clearly diminished. Thus, although the interactions of molecular forceps with peptides are not crucially dependent on structural preorganization, receptors with a rigid design are clearly superior to flexible molecular forceps.     

Expression of the Fc receptor for IgG (Fc gamma RII/CDw32) by human circulating T and B lymphocytes

Tissue-specific isoforms of the human FcR for IgG Fc gamma RII (CDw32) have previously been described by using mAb. These mAb were shown to exhibit different patterns of reactivity with lymphocytes. Among human PBL, Fc gamma RII has been detected on B cells but not T cells when assessed by flow cytometry and microscopy with the use of mAb KB61 and 41H16. Although KB61 and 41H16 were found to react with B cells, the mAb IV.3, CIKM5, and 2E1 did not react with any PBL subset. In this study, we show that KB61 and 41H16 react strongly with the majority (93-96%) of B cells (CD20+), and weakly with a proportion (18-42%) of T cells (CD3+), including 10 to 14% of CD4+ and 27 to 69% of CD8+ cells. In addition, mRNA for Fc gamma RII was detected in purified CD3+CD8high+ lymphocytes by polymerase chain reaction. KB61 and 41H16 also reacted with a majority of CD3-CD16/CD56+ cells, and CD3-CD20- cells. These findings indicate that a subset of T cells and non-T/non-B cells express Fc gamma RII, and are of interest in the light of previous studies which postulate that human Fc gamma R+ cells and Fc gamma RII+ murine T cells suppress the B cell immune response.     

CD3 hyporesponsiveness and in vitro apoptosis are features of T cells from both malignant and nonmalignant secondary lymphoid organs

There is a dogma in tumor immunology that tumor-infiltrating lymphocytes (TIL) are defective based on their lack of antitumoral efficacy in vivo and on impaired response to in vitro functional tests. However, TIL have been compared usually with peripheral blood T lymphocytes, raising doubts on the conclusions drawn. Therefore, we compared TIL from B cell non-Hodgkin's lymphomas (NHL) with T cells from nonmalignant secondary lymphoid organs. NHL-TIL were unresponsive to activation by immobilized anti-CD3 mAb, although bypassing T cell receptor (TCR)/CD3 signaling led to proliferation. The poor proliferative responses of NHL-TIL could not be explained by quantitative defects in TCRzeta expression. NHL-TIL underwent marked spontaneous apoptosis in vitro with loss of approximately 50% of cells after 24 h of culture. This was associated with downregulation of the antiapoptotic Bcl-xL and Bcl-2 proteins, whereas viable NHL-TIL maintained their expression. IL-2, anti-CD3/IL-2, and manipulation of the Fas/Fas-ligand death pathway had no effect on NHL-TIL survival. Apoptosis was not due to increased cell cycling, as NHL-TIL were quiescent, nonproliferating cells. T cells from inflammatory, nonmalignant tissues gave similar functional results to NHL-TIL, suggesting the existence of factors common to the microenvironment of these diverse pathologies. Thus, the quiescent, anergic phenotype of NHL-TIL cannot be attributed solely to tumor factors, but rather is a feature of T cells from chronic inflammatory lesions.     

Regulation of alphaIIb beta3 function in human B lymphocytes

We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.     

Enteral nutrition modifies gut-associated lymphoid tissue in rat regardless of the molecular form of nitrogen supply

OBJECTIVES: It is not known whether the results of randomized trials comparing coronary artery bypass grafting to percutaneous transluminal coronary angioplasty for initial revascularization apply to repeat revascularization in patients with prior bypass grafts. We studied the differences between the patients with prior bypass grafts referred for surgery or angioplasty to identify the clinical and angiographic characteristics that correlated best with either choice and to find clues that might aid in selecting one treatment over the other. METHODS: Between 1992 and 1994, 870 patients underwent first isolated reoperation and 793 patients underwent first balloon angioplasty after a previous operation. A jeopardy score (0 to 8 points) was derived for each patient on the basis of the relative size of the ischemic territory. Clinical and angiographic data were analyzed for association with the revascularization strategy. RESULTS: The following characteristics were more prevalent in the reoperation group: male sex, diabetes, hypertension, valvular disease, normocholesterolemia, and severe left ventricular systolic dysfunction; fewer functioning venous and arterial grafts; and a higher jeopardy score (p < 0.01 for all) than in the angioplasty group. A higher jeopardy score, diabetes, and a lower number of functioning arterial or venous grafts were strong, independent predictors of referral for reoperation (p < 0.01 for all). In hospital death and Q-wave infarction (p < 0.01 for both) were more frequent in the reoperation group. CONCLUSIONS: Reoperation was the revascularization procedure of choice when larger regions of myocardium were in jeopardy. Angioplasty was more frequently chosen in the presence of a patent arterial graft to the left anterior descending coronary artery or multiple functioning bypass grafts. Reoperation was associated with a higher risk of in-hospital complications than angioplasty.     

Alteration of signal-transducing molecules in tumor-infiltrating lymphocytes and peripheral blood T lymphocytes from human colorectal carcinoma patients

An analytical method for the rapid isolation and recovery of the homologous series of 2-aminoethanols, a class of organic compounds of importance to wood preservative treatment, is successfully developed. The method is applied to an aqueous solution of copper amine (copper[II] hydroxide complexed monoethanolamine) and to copper-amine-treated sawdust. The method incorporates a gas chromatograph-ion-trap mass spectrometer. A discussion of the secondary equilibrium effects involved when ionizable analytes are extracted from an aqueous phase with respect to organic bases is presented. Using 2-propanol as the extractant coupled to a salt-saturated aqueous phase results in recoveries of 63% for 2-aminoethanol, 51% for N,N-dimethyl-2-aminoethanol, and 56% for N-methyl-2-aminoethanol for a single liquid-liquid extraction. The choice of 2,2,2-trifluoroethanol as an internal standard is found to be quite suitable. A comparison of the precision and accuracy for an external versus an internal mode of instrument calibration demonstrates that the internal standard mode is preferable for this manual injection.     

cis-dominant regulation of CD4 and CD8 gene expression in rat/mouse T cell heterohybridomas

To determine whether expression of CD4 and CD8 molecules on T cells is determined-solely by transacting regulators, we examined heterohybridomas derived from the fusion of a rat CD4+ T cell line and the CD4- CD8- mouse thymoma BW5147. The majority of hybrid offspring expressed rat CD4. However, a fraction of the cell lines obtained expressed not only rat CD4 but also various amounts of mouse CD4 and CD8 molecules from both species. Cloning of two of these heterogeneous lines revealed that expression of all four Ag varied not only between different clones but also within clonal populations. The expression of Ag not present on the parental cells suggested an alteration in the normally stable regulatory mechanisms present in those cells. Moreover, a lack of concordant expression between the rat and mouse loci was observed, indicating that active and silent homologous loci can exist together in single nuclei. Expression of CD4 and CD8 in these cells, therefore, cannot be solely mediated by trans-acting diffusible regulators but must also depend on cis-dominant effects on the loci themselves. The phenotypic heterogeneity of clonal populations was found to result from unpredictable shifts, both positive and negative, in the expression of CD4 and CD8 over time, indicating that the cis-dominant effects were only quasistable. Preliminary examinations of the density of 5-methylcytosine within the CD4 and CD8 loci in various phenotypic populations separated by FACS from within heterogeneous clones revealed a correlation between surface expression of the mouse CD8 protein and a lack of methylation around the mouse CD8 gene. In contrast, the CD4 gene remained extensively methylated regardless of its surface expression.     

Enhanced generation of cytotoxic T lymphocytes using recombinant vaccinia virus expressing human tumor-associated antigens and B7 costimulatory molecules

In this work, we addressed the possibility to enhance the "in vitro" generation of CTLs recognizing tumor-associated antigens (TAAs) by using an inactivated recombinant vaccinia virus encoding B7.1 and B7.2 costimulatory molecules (rVV-B7.1/2). Antigen presenting cells (APCs) infected by rVV-B7.1/2 and pulsed with MART-1/Melan-A27-35 HLA-A2.1-restricted peptide induced significantly higher specific cytotoxic activity than peptide-loaded APCs infected by wild-type VV, both in VV-sensitized and naive donors. When APCs were infected with a rVV encoding both MART-1/Melan-A27-35 and B7-1/2 (rVV-B7.1/2-M), a significantly more effective CTL generation was observed as compared with cultures stimulated by APCs infected with a rVV encoding the TAA epitope only (rVV-M). These enhancing effects were detectable irrespective of a previous VV-specific sensitization. Most importantly, fibroblasts, devoid of antigen-presenting capacity upon peptide pulsing or infection with rVV-M, could be turned into effective APCs after infection by rVV encoding TAA epitopes and costimulatory molecules. In these experiments, by using separate recombinant viral constructs, we observed a predominant role of B7-1 as compared with B7-2 in the induction of TAA-specific CTLs. Taken together, our data indicate that replication-incompetent rVV encoding TAA epitopes and costimulatory molecules are able to induce highly effective generation of tumor-specific CTLs. Therefore, these vectors could represent valuable clinical tools for immunotherapy of melanoma patients.