The apical submembrane cytoskeleton participates in the organization of the apical pole in epithelial cells.

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145-3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40-70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical-basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37 degrees C or in n-octyl-beta-D-glycoside at 4 degrees C (representative of GPI-anchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+-K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.

Neonatal transplantation tolerance is associated with a systemic reduction in memory cells, altered chimeric cell phenotype, and modified eicosanoid and cytokine production.

Certain B10 background mice are resistant to tolerance induction following a neonatal inoculation of semiallogeneic class I/II MHC-disparate cells despite early thymic clonal deletion of alloreactive cells. The emergence of memory T cells and persistence of particular chimeric cells in the thymus has an association with this resistance. In these studies, we utilized a hemisplenectomy technique to examine systemic cell populations of adult Bl0.S (H2s, H2E-) mice that received (Bl0.S x B10.A)F1 cells at birth and before and following application (and rejection or acceptance) of Bl0.A (H2k/d, H2E+) skin grafts. Prior to skin graft challenge, tolerant mice had reduced splenic levels of memory (CD45hi, PgP-1hi, Mel-14neg) T cells as compared with the rejecting recipients and following B10.A graft challenge, the nontolerant mice showed a further increase in these cells. Elevated pretransplant levels of donor H2Kk+ cells coexpressing B220, CD11b, or CD3 were seen in the tolerant mice. Following skin grafting, splenic chimerism was reduced with differing chimeric cell phenotypes between the tolerant and nontolerant mice. In vitro production of PGE2 in a MLC was delayed in the tolerant mice with minimal production of IL-2 and IL-4. Nontolerant mice made high levels of TxB2 and heightened, early production of IL-2 and IL-4 during the MLC. Thus, tolerance induction is associated with increased numbers of particular chimeric cells, fewer peripheral lymphoid immunocompetant memory T cells, impaired eicosanoid secretion, and reduced alloreactivity and alloantigen-driven IL-2/IL-4 production. It appears that alloreactive cells necessary to break tolerance are generated when fewer class II+ (e.g., B220+, CD11b+) chimeric cells are present and that there is a coexistence of effector and regulatory T cell subpopulations in the nontolerant mice. By comparison, tolerance acquisition does not appear associated with the presence or generation of a predominant subtype of T cell but rather is likely more dependent upon clonal deletion processes.

Destructive allograft fungal arteritis following simultaneous pancreas-kidney transplantation.

Fungal arteritis of the Y graft used to revascularize the whole pancreas graft developed in 2 recipients of simultaneous pancreas-kidney transplant that were performed within 36 hr of each other. The vascular infection became manifest 6-7 days following transplantation. In both patients, the vasculitis culminated in an arterial rupture that required immediate operative intervention. This compromise of the Y grafts contributed to loss of both pancreatic grafts and necessitated vascular reconstruction to reperfuse the lower extremity. To date, both patients continue to experience normal kidney transplant function.

Longitudinal induced IL-2 mRNA monitoring in renal transplant patients immunosuppressed with cyclosporine and in unmodified canine renal transplant rejection.

Immune monitoring of transplant patients to define optimal immunosuppression continues to be important, as rejection occurs despite adjustment of dosaging of CsA or even FK506 to achieve "therapeutic-range" blood levels. Because CsA is known to inhibit upregulation of IL-2 mRNA transcription, we prospectively sequentially measured (induced) IL-2 mRNA in PHA-stimulated PBMC cultures from transplant recipients of kidneys from living-related donors (n = 15) using a quantitative PCR assay, with a potential 24-hour turnaround time, to define immunologic events in real time. Reproducible individual patient sensitivity or refractoriness to CsA was determined pretransplant, by adding a range of CsA concentrations to the PBMC cultures and constructing induced IL-2 mRNA regression inhibition curves. However, this was not predictive of rejection episodes, but did correlate well with individual differences in IL-2 mRNA levels posttransplant, despite similar maintenance trough blood concentrations of CsA between patients. In this prospective study, seven patients experienced rejection episodes despite therapeutic CsA trough levels. Three of these, plus one not receiving CsA therapy, who happened to be prospectively tested at the time that rejection was clinically diagnosed, had a decrease in induced IL-2 mRNA before treatment was instituted. As a correlation to this observation in patients, induced IL-2 mRNA levels in unmodified rejection were sequentially measured in PBMC cultures in autologous vs allogeneic canine renal transplants and IL-2, IL-10, TNF-alpha, and IFN-gamma mRNA were also measured in kidney biopsies. Sequential PHA lymphoproliferation assays of [3H] thymidine incorporation on patient and dog PBMC cultures were also performed. Similar to the observations in patients, unmodified rejection in the canine renal allograft model also was accompanied by a decline of PHA-induced IL-2 mRNA in PBMCs as the serum creatinine concentrations became elevated. In the dog kidney biopsies at later phases of rejection, IL-10 mRNA levels were also significantly elevated (p = 0.032).

Malassezia furfur--disseminated infection in premature infants.

Three infants, born prematurely, died after clinical illnesses of 67, 65, and 60 days from infection by Malassezia furfur. Each infant had been nourished with lipid emulsions delivered through deep-line catheters. The infections, all discovered at autopsy, were characterized by massive involvement of lungs. Two of the three had endocardial vegetations containing M. furfur; all three had lesions in liver, kidney, and spleen, and two had lesions in adrenal, pancreas, and colon. In addition, one of the infants had acute meningoencephalitis caused by M. furfur. In some of the distant organs, yeast cells of M. furfur were growing in the lumina of small vessels, filling the lumina, but causing no vasculitis or infarction. In addition to these benign collections of yeasts within vessels, there were acute inflammatory lesions as well. These were consolidation, vasculitis, granulomatous inflammation, septic thrombosis, and septic infarction of lung and foci of necrosis and inflammation in kidney and liver. Two previously reported autopsies described neonates with lesions in lung and heart. The authors' three cases for which autopsies were performed had lesions in lung and heart too but, in addition, had dissemination with acute lesions in kidney and liver. Finally, one patient had a severe meningoencephalitis caused by M. furfur.

Differential patterns of T cell clonal deletion in neonatal H-2 tolerance and I-E/Mls induced self-tolerance.

The pattern of clonal deletion of putative I-E-reactive (V beta 11) and Mls-reactive (V beta 3) T cells was evaluated and compared by cytofluorographic and immunohistochemical methods in a model of neonatal H-2 tolerance and in I-E- or Mls-bearing strains of mice which normally delete these cell populations (self-tolerance). The ontogeny of deletion of V beta 11+ cells was studied by evaluating thymic changes from birth until maturity in B10.S (H-2s/I-E-), B10.A (H-2k/d/I-E+) and B10.S mice intravenously infused at birth with (B10.SxB10.A)F1 lymphohaematopoietic cells. The reduction in V beta 11+ cells was most prevalent in the medullary region of the naive B10.A and neonatally injected B10.S animals and was corroborated by flow cytometry which demonstrated a marked reduction in single CD4 and CD8 positive B beta 11 T cells when compared to naive B10.S mice. However, immunohistochemistry illustrated that 'deletion' was never complete since V beta 11+ cells remained in the thymic cortex and splenic lymphoid follicles. By comparison, DBA/2 mice (Mlsc+ and previously documented to have decreased levels of V beta 3+ cells) showed a different pattern of deletion of V beta 3+ T cells than what was found for T cells bearing V beta 11 in animals deleting this population. DBA/2 thymi contained fewer thymic V beta 3- cells and there was more complete elimination of these cells, particularly in the periphery, by flow cytometry and immunohistology. The mice which do not delete V beta 3 cells (Mlsc-) showed that the majority of V beta 3- cells were located in the medulla with a few cells distributed in the cortical region. This pattern was notably different than the distribution of V beta 11 cells in thymi. Despite their location by histology, the majority of remaining V beta 3+ cells were dual CD4/CD8 positive (CD4+CD8+) by flow cytometric analysis. Our data illustrate that V beta 11 and V beta 3 T cells appear to be eliminated (i.e. 'deleted') at similar stages of maturation (single positive) during self-tolerance as well as in a neonatal H-2 tolerance model. However, the degree of elimination and the location of the cells remaining in these mice is dramatically different, depending on which T cell population is being evaluated and which deleting ligand is presented intrathymically. Thus, the accepted tenet of dual CD4+CD8+ cells localizing to the thymic cortex appears to have exceptions.(ABSTRACT TRUNCATED AT 400 WORDS)

Dipeptidyl peptidase IV (CD26) activity in human alloreactive T cell subsets varies with the stage of differentiation and activation status.

Dipeptidyl peptidase IV (DPP IV), also known as CD26, is a transmembrane serine aminopeptidase which has an ontogenically related expression on T cells and participates on several immunological functions. CD26 appears to play an important role in alloimmunity during host T cell activation subsequent to alloantigen encounter and is a way by which effector T cells traverse graft endothelial barriers. In order to help to elucidate the role of the CD26 molecule in alloimmune responses, DPP IV activity and CD26 antigenic expression were assessed during the initial phases of completely MHC-disparate human mixed lymphocyte reactions (MLRs) and in several long-term alloreactive T cell clones. Our methods involved the use of a rhodamine-110-conjugated dipeptide substrate specific for DPP IV in two-colour cytofluorographic analysis that allowed stimultaneous lineage marker evaluation. Polyclonal populations of alloreactive CD4 and CD8 T cells contained DPP IV activity at 1 and 10 min of incubation that was variably elevated from resting T cells with the enzyme activity confined to CD26+ cells. T cell clones derived from MLRs were established with IL-2 supplementation and alloantigen restimulation and had reduced CD62L expression with functional specificity to the stimulating MHC. While CD26 expression remained stable, DPP IV activity was variable in the alloreactive T cell clones, with enzyme function in the latter appearing to coincide with the timing of alloantigen restimulation. These studies demonstrate that DPP IV activity varies among phenotypically distinct alloreactive T cell subsets and appears to be altered with the activation status of the effector cells. These findings raise the potential of a role for CD26/DPP IV in the generation of specific alloimmunity. With this methodology, it may be possible to reveal whether specific alterations in the activity of this molecule in T cell populations promote graft acceptance and to determine the molecular requirements for these changes.

Pulmonary embolism with bone fragments following vertebral body marrow infusion for tolerance induction.

Protocols of donor bone marrow infusion for tolerance induction are receiving increasing attention in clinical trials of organ allotransplantation. We report pulmonary embolism with bone fragments following vertebral body marrow infusion in a recipient of a liver and intestinal transplant. Even though pulmonary embolism with bony microfragments has been widely described following bone marrow transplantation, the use of single, high-dose donor bone marrow infusion and/or multiple infusions currently under clinical investigation for induction of donor specific unresponsiveness, may warrant the implementation of additional steps in the vertebral body marrow processing technique to decrease or eliminate the component of bony microfragments in the final preparation.

Diagnosis of intraocular lymphoma by flow cytometry.

PURPOSE: To evaluate flow cytometry of vitreous cellular specimens as a means of diagnosing intraocular lymphoma and ocular inflammatory disease. METHODS: We undertook a retrospective, observational study of hematopoietic cell-surface markers in 20 patients with vitreous cellular infiltration in whom lymphoma was considered in the differential diagnosis. Immunophenotyping of vitreous cells obtained by vitrectomy was performed by flow cytometry using antibodies directed against specific cell-surface antigens, including ones associated with B-lymphocyte and T-lymphocyte lymphomas and activated inflammatory cells. Smears were examined cytologically. Cytofluorography was compared with the cytopathologic diagnosis and with final diagnosis. RESULTS: With flow cytometry, a diagnosis of intraocular lymphoma was confirmed in two of four patients with known lymphoma, one of whom had recurrent disease after radiation, and not confirmed in two patients who had had prior treatment with radiation or corticosteroids. In six patients with no prior diagnosis of lymphoma, five were diagnosed with lymphoma on the basis of cytofluorography. Thus, seven (70%) of 10 patients with intraocular lymphoma were diagnosed by cytofluorography compared with three (30%) of 10 with lymphoma diagnosed by cytology. With flow cytometry, 10 patients with uveitis or intraocular infections were distinguishable from patients with lymphoma by lack of a monotypic population and, in some cases, by elevated CD4:CD8 ratios and a high percentage of activated cells. CONCLUSIONS: Cytofluorography of vitreous cells is an effective alternative or adjunct to cytology. Information can be gained from specimens that are uninterpretable by routine cytology. The optimal technique for diagnosis may vary among institutions.

Fecapentaenes and their precursors throughout the bowel--results of an autopsy study.

The fecapentaenes are potent mutagens found in the stool of some humans and pigs. These compounds are produced by Bacteroides species in the gut from an uncharacterized family of precursor compounds, and have been postulated to pose a risk of human colorectal cancer. To better understand fecapentaene production in vivo, and to determine if excreted levels measured in epidemiologic studies are representative of the entire colon, fecapentaenes were assayed from multiple sites in the bowel in an autopsy study of 16 humans and 2 pigs. An indirect measurement of fecapentaene precursors was also made. Colonic concentrations of fecapentaenes and precursors varied widely between individuals, but were consistent for each individual throughout the colon. In addition, the measurements of rectal contents, assumed to approximate values in excreted stool, were equivalent to measurements from the colon.

Cytofluorographic evidence that thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny and apoptotic status.

CD26 is a multifunctional molecule implied to have a variety of roles in the immune response including its activity as a membrane exopeptidase (Dipeptidyl peptidase IV) which cleaves several protein molecules. In order to further define the expression and functional activity of CD26 in the developing thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of DPP IV activity with a fluorochrome-conjugated peptide substrate and surface staining of the T lymphocyte lineage antigens CD4 and CD8. Neonatal and adult murine thymi were examined using the three-color assay and significant differences in DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. Single-positive cells bore higher activity than CD4-/CD8- cells and neonates had higher activity than adults. Thymocytes with characteristics consistent with apoptotic cells expressed higher DPP IV activity. Thus, DPP IV appears to be upregulated both as thymocytes mature and among thymocytes which are undergoing programmed cell death. These results suggest that CD26 is ontogenically controlled during T cell maturation and may play a role in thymic deletion of emerging clones.

Simultaneous expression of CD2 on a B-cell, small lymphocytic neoplasm.

An unusual neoplasm, best classified as a B-cell chronic lymphocytic leukemia on the basis of cytofluorographic, immunohistologic, and gene rearrangement analysis also co-expressed the T-cell associated marker CD2 (sheep erythrocyte receptor), but without other cell markers restricted to T cells. This case was associated with a more aggressive course than typically seen with B-CLL or malignant lymphoma, small lymphocytic type, implying along with previous reports that CD2 expression may serve as a marker of small lymphocytic tumor cell behavior and the ultimate clinical course. Thus, surface phenotypic analysis of these particular hematopoietic neoplasms may serve not only for identification of the malignancy, but could also render prognostic information.

DNA flow cytometric measurements in breast cancer: a review of the instrumentation principles and clinical significance.

Breast cancer treatment remains a controversial and challenging issue. Treatment of this disease is often guided by the clinical stage and type of tumor present, as well as information provided by a variety of prognostic indicators which can be employed. DNA analysis by flow cytometry is a relatively new technique that is emerging as a powerful tool to predict the biologic behavior of breast tumors. A majority of studies have demonstrated that the S-phase fraction measured by DNA flow cytometric is an independent predictor of patient outcome. There is less conclusive information regarding the association of DNA ploidy with the clinical behavior of breast malignancies, although, tumor grading often closely parallels this DNA parameter. New flow cytometry methodology involves multiparameter analysis that allows a simultaneous examination of DNA ploidy and cell cycle analysis with surface or internal molecules. This newer approach permits a precise delineation of DNA characteristics in distinct cell populations within a tumor and will likely enhance the utility of this procedure in determining the biologic behavior of the breast tumor.

Borderline tuberculoid Hansen's disease in AIDS.

We performed an immunohistochemical analysis of a skin lesion from a patient with AIDS who had borderline tuberculoid Hansen's disease. We also evaluated other laboratory features and performed peripheral blood flow cytometric analysis. The in situ immunologic response to Mycobacterium leprae was minimally affected by concomitant infection and immunosuppression by HIV. The skin demonstrated the typical characteristics of borderline tuberculoid lesions. These results indicate that although a patient with HIV infection may have laboratory evidence typical of the immunosuppression seen in AIDS, the immunologic response to M. leprae is essentially unchanged.

Characterization of donor chimerism, alloreactive host T cells and memory cell development in thymi from mice resistant to neonatal transplantation tolerance.

Certain B10 background mouse strains are resistant to tolerance induction after neonatal inoculation of class I/II MHC-disparate F1 hybrid cells. Despite initial thymic deletion of alloreactive cells, the majority (95%) of these mice reacquire the capacity to reject donor allografts. These current studies examined thymi of adult B10.S (H-2S/H-2E-) mice that neonatally received (B10.S x B10.A)F1 cells, before and after rejection or acceptance of B10.A (H-2k/d/H-2E+) skin grafts. Alloreactive thymic V beta 11+ and V beta 5+ T cells were often reduced in the injected recipients preceding allograft challenge. In some mice a single B10.A skin graft generated an increase in these T cell populations concurrent with allograft rejection. Injected mice that accepted B10.A skin grafts (i.e., tolerant) or that rejected two sequential B10.A grafts often had a reduction of these T cells. Thymic memory cells (CD44high+) were present before transplant in injected mice and reduced in the tolerant mice. Donor chimeric cells were identified in the majority of injected mice before transplant and diminished after B10.A graft application. A greater proportion of chimeric cells coexpressed CD11b or B220 in the tolerant mice. Thus, neonatally injected B10.S mice resistant to tolerance induction reacquire immunocompetent thymic T cells bearing characteristics of memory T cells that could mediate graft rejection. Finally, the initial presentation of chimeric cells (e.g., macrophages) that may efficiently present class I Ags through H-2E likely increases the possibility of adult tolerance.

Human thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny.

The nonintegrin receptor CD26, also known as dipeptidyl peptidase IV (DPP IV) is a transmembrane 110- to 120-kDa serine aminopeptidase glycoprotein with multiple functions, including cellular trafficking through extracellular matrix, and costimulatory potential during T cell activation, and is an influence upon T cell differentiation during their maturation in the thymus. In order to further define the expression and functional activity of this membrane exopeptidase in human thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of intracellular DPP IV activity using a fluorochrome-conjugated peptide substrate with surface staining of plasma membrane-associated T lymphocyte lineage antigens CD4 and CD8, as well as CD26. Human thymi were examined using the three-color assay, and significant differences in time-dependent DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. In this regard, CD4(-)/CD8(-) thymocytes displayed the lowest DPP IV activity and had higher activity than the smaller-sized CD26(+) cells. Thymocytes containing greater percentages of apoptotic cells expressed lower DPP IV activity than viable cells. Thus, DPP IV appears to be upregulated as thymocytes mature and is reduced among thymocyte populations enriched for cells undergoing programmed cell death, suggesting that CD26-associated enzymatic activity is ontogenically controlled during T cell maturation and may be involved in thymic deletion of emerging clones.