Monitoring of donor/recipient T-cell engraftment kinetics in myeloablative allogeneic stem cell transplantation using short tandem repeat amplification from cell lysates.

Molecular monitoring of donor/recipient T-cell kinetics early post-transplant can provide clues to the immunological events that govern host-versus-graft reaction (HVGR) and graft versus-host-disease (GVHD). We have previously used fluorescence in situ hybridization (FISH) with X and Y probes to monitor recipient T (R-T) cell clearance early after myeloablative allogeneic stem cell transplantation (ASCT). We demonstrated that impaired clearance of residual host-T-cells in the early days post-transplant was associated with graft rejection, while enhanced clearance could be an indicator of increased donor anti-host alloreactivity and predictive of acute GVHD. Although FISH is the most accurate quantitative molecular tool for the determination of the exact donor/recipient-T-cell numbers at any time points post-transplant, it has the disadvantage of being limited to sex mismatched donor/recipient pairs. Our goal was to develop a molecular approach that, irrespective of gender, would be comparable to FISH in accurately determining host residual T-cell clearance after myeloablative conditioning for ASCT. We have genotyped DNA from cell lysates using polymerase chain reaction (PCR) amplification of short tandem repeats (STR) with fluorescently labeled oligonucleotide primers, and used the Genescan 672 software for accurate quantitative analysis of the amplified alleles. Here, we show that this approach allowed us to achieve in T-cells accurate quantitative analyses of amplified donor/recipient alleles in sex matched patients on days +5, +8 and +12 post-transplant, despite severe leukopenia.

Immunophenotyping of acute leukemia at King Faisal Specialist Hospital and Research Centre.

During a 20 month period, 133 bone marrow samples from an equal number of patients with acute leukemia were immunophenotyped. Patients ranged in age from two to 68 years with a mean of 23 years. Eighty-four (63.2%) were classified as acute lymphocytic leukemia (ALL) with the following immunologic subclassification: common ALL 83.3%, T-cell ALL 11.9%, null-cell ALL 2.4% and 2.4% differentiated B-cell ALL. Twenty-eight cases (21%) were classified as acute myeloid leukemia (AML) and 16 cases (12%) demonstrated biphenotypic features. Concordance with morphology and cytochemistry was observed in 129 cases (97%). Four cases (3%) manifested discrepancy between immunophenotyping, morphology and cytochemistry. We conclude that immunophenotyping by flow cytometry is a useful and reliable method for classification of acute leukemia, especially when interpreted in the light of morphology and cytochemistry.

Immunophenotyping of childhood acute lymphoblastic leukemia in Saudi Arabia: second look.

Geographical variations in the incidence of disease are of considerable theoretical and practical importance. It has been claimed that the distribution of acute lymphoblastic leukemia (ALL) phenotypes in Saudi Arabia is different from that recorded in the Western literature. One hundred and twelve (112) patients under 15 years of age, diagnosed as ALL between January 1992 and May 1994 had immunophenotypes performed on their blast cells. Common ALL (cALL) together with pre-B-ALL, formed 86.5% of the total; B-cell 3%, T-cell 6% and null cell 4.5%. These figures are not significantly different from the Western literature. A previous claim from this institution in 1990, that both null and B-cell ALL were significantly increased compared with elsewhere, is not supported by the present figures. Age and sex distribution, and FAB classification, L1 77%, L2 20% and L3 3%, were also of the same order as described elsewhere and, in particular, there was no increase in the frequency of L3 subtype.

Reaction of the avian respiratory system to intratracheally administered avirulent Salmonella typhimurium.

Chickens were inoculated intratracheally (IT) with the SR-11 Salmonella typhimurium deletion mutant x4062 strain. Data collected for 8 days postinoculation (PI) were: signs of respiratory and gastrointestinal disease; histological lesions; the influx, phagocytic proportion, and phagocytic capacity of avian respiratory phagocytes (ARPs); and the proportion of granulocytes vs. macrophages in the lung tissues and lavage fluids of the lungs and air sacs. S. typhimurium-inoculated chickens had no clinical signs of gastrointestinal or respiratory disease but had various degrees of inflammatory changes in the lungs. At 5 hr PI, S. typhimurium-inoculated chickens had approximately 53-fold more ARPs than mock-inoculated controls. Between 26 hr and 8 days PI, the number of ARPs from S. typhimurium-inoculated birds was not significantly higher than the number from the mock-inoculated controls. Flow cytometric analysis of ARPs demonstrated that the proportion of phagocytic ARPs and the phagocytic capacity of ARPs from S. typhimurium-inoculated chickens were significantly higher between 5 and 26 hr PI than those of the ARPs from mock-inoculated chickens. Kinetic changes over 8 days in the granulocyte/macrophage ratios in the lavage fluids, as compared with kinetic changes in the lung tissues, suggested that the granulocytes generally represent a much higher proportion of the ARPs, and egress earlier and in much larger numbers from the tissues to the lumen of lungs and air sacs than do macrophages.

Lack of formyl-methionyl-leucyl-phenylalanine receptors on porcine neutrophils.

The response of blood neutrophils to the chemotactic peptide formyl-methyl-leucyl-phenylalanine varies among species. Our results indicate that this peptide does not activate the respiratory burst of porcine neutrophils. Specifically, concentrations less than or equal to 10(-6) M did not cause production of either superoxide or hydrogen peroxide. Studies designed to delineate the biochemical deficit responsible for these results indicated that these cells do not express specific chemotactic peptide receptors on the external surface of the plasma membrane. Although these data do not rule out the possibility that internal stores of chemotactic peptide receptor exist, attempts to induce expression of the receptor by priming the cells with either lipopolysaccharide or calcium ionophore were unsuccessful.

Evidence for major alterations in the thymocyte subpopulations in murine models of autoimmune diseases.

Thymocytes can be divided into four major subpopulations: CD4+CD8+ (double-positive), CD4-CD8- (double-negative), CD4+CD8- (CD4+) and CD4-CD8+ (CD8+) cells. Recent studies have shown that T-cell development in the thymus progresses as: CD4-CD8(-)----CD4+CD8(+)----CD4+ or CD8+ cells. In the present study we investigated these and other subpopulations of thymocytes in autoimmune MRL(-)+/+, MRL-lpr/lpr, C57BL/6-lpr/lpr, BXSB and NZB mice before (1-month old) and after (4-6-months old) the onset of lymphadenopathy and autoimmune disease. All the autoimmune strains at one month of age and other H-2, sex and age-matched controls (C3H, DBA/2, and C57BL/6) demonstrated normal proportions of thymocyte subsets with approximately 75% double-positive cells, 5-7% double-negative cells, 11-15% CD4+ cells and 3-5% CD8+ cells. By 4-6 months of age, MRL(-)+/+ mice demonstrated a moderate increase in double-negative cells (approximately 13%) and a decrease in double-positive cells (approximately 46%). Interestingly, in the presence of the lpr gene, as seen in MRL-lpr/lpr mice, the double-negative cells increased to approximately 47% and the double-positive cells decreased to approximately 16%. In contrast, 4-6-month-old C57BL/6-lpr/lpr mice failed to demonstrate any alterations in the thymocyte subsets thereby suggesting that background genes, in addition to the lpr gene, played a role in the thymocyte differentiation. BXSB male mice with severe lymphadenopathy behaved very similarly to MRL-lpr/lpr mice, inasmuch as their thymus contained approximately 48% double-negative cells and only approximately 8% double-positive cells. In contrast to MRL-lpr/lpr and BXSB strains, NZB mice at 6 or 10 months of age had normal composition of thymocyte subsets. In MRL and BXSB animals, although there was a significant increase in CD4+ cells (approximately 23-33%), due to a consequent increase in CD8+ cells (approximately 11%), the ratio of CD4+:CD8+ cells remained 2-3:1, similar to that seen in normal mice. Furthermore, using the J11d marker expressed by the majority of the double-negative and all double-positive thymocytes but not by mature functional T cells, we confirmed the above findings and demonstrated further that MRL-lpr/lpr mice at 4-6 months of age had an increased percentage of J11d- double-negative cells and a decrease in J11d+ double-negative cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in macrophage populations: phenotypic differences between normal and tumor-bearing host macrophages.

As a tumor grows, changes occur in the function of macrophages (M phi). This is concomitant with changes in their phenotype. Flow cytometric analysis of monoclonal antibody (mAb)-labeled thioglycollate-elicited peritoneal, and resident splenic, M phi showed a tumor-induced shift of Mac-1, -2, -3, and Ia antigen expression. During tumor growth, the percentage of peritoneal Mac-2+, -3+, and Ia+ M phi decreased significantly (22%, 14%, and 58%, respectively), while Mac-1+ M phi remained unchanged. By analyzing the data on two-dimensional histograms and comparing the sizes of M phi to cell-surface antigen expression, we identified distinct subpopulations of peritoneal M phi. Three distinct size versus antigen expression M phi subpopulations were detected by flow cytometry and consisted of 10-16, 17-22, and 23-27 microns for the small-, medium-, and large-sized populations, respectively. Large-sized Mac-1+ and -2+ M phi decreased (37% and 38%), while large-sized Mac-3+ M phi did not decrease during tumor growth. Medium-sized Mac-3+ M phi decreased 33% during tumor growth, while no differences could be seen in medium-sized Mac-1+ or -2+ M phi. Concomitant with the decrease in large-sized Mac-1+ M phi was an increase in small-sized Mac-1+ M phi. Peritoneal Ia+ M phi were mostly small-sized (4-7-fold increase over the medium-sized and none in the large-sized population). M phi Ia antigen expression was nearly absent in the 21-day tumor-bearing host, with less than 4% of the cells labeling positive (a 73% drop from normal host M phi). In splenic M phi, the percentage of Mac-1+ M phi significantly increased (90%) during tumor growth, while Mac-2+ and -3+ M phi showed a smaller, but still significant, increase (48% and 40%, respectively). Additionally, splenic Ia+ M phi significantly decreased (29%) during tumor growth. More important than the decreased cell numbers was the significant decrease in Ia antigen expression per cell. Unlike the peritoneal M phi, the splenic M phi did not show distinct size versus antigen expression subpopulations, although there was an overall difference in M phi size between normal and TBH. These data suggested that M phi from different anatomical sites are phenotypically different and tumor growth mediates phenotypic alterations in peritoneal and splenic M phi populations. This may be the source of tumor-induced dysfunction of M phi-mediated immune activity.

Cellular defense of the avian respiratory system: effects of Pasteurella multocida on respiratory burst activity of avian respiratory tract phagocytes.

The respiratory tract of healthy chickens contain few free-residing phagocytic cells. Intratracheal inoculation with Pasteurella multocida stimulated a significant (P less than 0.05) migration of cells to the lungs and air sacs of White Rock chickens within 2 hours after inoculation. We found the maximal number of avian respiratory tract phagocytes (22.9 +/- 14.0 x 10(6] at 8 hours after inoculation. Flow cytometric analysis of these cells revealed 2 populations on the basis of cell-size and cellular granularity. One of these was similar in size and granularity to those of blood heterophils. Only this population was capable of generating oxidative metabolites in response to phorbol myristate acetate. The ability of the heterophils to produce hydrogen peroxide, measured as the oxidation of intracellularly loaded 2',7'-dichlorofluorescein, decreased with time after inoculation. These results suggest that the migration of heterophils, which are capable of high levels of oxidative metabolism, to the lungs and air sacs may be an important defense mechanism of poultry against bacterial infections of the respiratory tract.

Expression of the J11d marker on peripheral T lymphocytes of MRL-lpr/lpr mice.

MRL-lpr/lpr (lpr) mice spontaneously develop massive lymphadenopathy resulting from the expansion of a unique population of Thy-1+ cells which are CD4- and CD8- (double negative) and the nature of which is not clear. The antibody J11d has been shown to define a differentiation Ag found on immature thymocytes but not on mature and functional peripheral CD4+ or CD8+ T cells. To analyze the possible relationship between the lpr double-negative T cells and the thymocytes, we investigated the simultaneous expression of J11d and Thy 1 Ag on the double-negative lpr lymph node cells by using two-color immunofluorescent staining technique. We observed that lpr mice at 3 to 4 weeks of age, before the onset of lymphadenopathy, did not have significant numbers (less than 4%) of J11d+ T cells in the periphery, similar to the number found in the control MRL +/+ mice. However, with increasing age of approximately 8 to 10 weeks and coinciding with the appearance of lymphadenopathy, a significant number (approximately 35%) of J11d+ Thy-1+ cells started appearing in the periphery of lpr mice and was maintained until the mice died at 20 to 24 weeks of age. The J11d+ T cells belonged to the abnormal double-negative T cell pool, inasmuch as J11d+ CD4+ or J11d+ CD8+ cells were absent in the lymph nodes of 20-wk-old lpr mice. Furthermore, 20-wk-old lpr mice demonstrated increased numbers (approximately 41%) of double-negative T cells in the thymus, a significant proportion of which were J11d+. In contrast, the 20-wk-old +/+ mice or 4-wk-old lpr mice had only 4% double-negative T cells in the thymus. The present study suggests that a significant number of peripheral double-negative T cells of lpr mice bear the immature thymic differentiation Ag J11d. The possibility that the accumulation of double-negative T cells results from abnormal peripheralization of double-negative J11d+ thymocytes, before complete differentiation into CD4+ or CD8+ T cells, is discussed.

Isolation of an antigenically "null" cell line from pig peripheral blood mononuclear cells.

A new pig cell line (A4) isolated from a primary culture of pig peripheral blood mononuclear cells was characterized. A4 was demonstrated to be morphologically, antigenically and functionally distinct from the more commonly isolated pig lymphoblastoid B cell lines (e.g. P-SC). When the A4 cell line and clones derived from it were tested against a panel of monoclonal antibodies, which define specific subpopulations of pig mononuclear cells, little or no reactivity was observed. The A4 cell line, unlike the P-SC cell line, was unable to induce a mixed lymphocyte reaction. The amount of immunoglobulin secreted by A4 cells as detected by an ELISA was reduced compared to that produced by P-SC cells. The P-SC cell lines produced an IL-1-like factor, whereas no IL-1-like activity was found in the A4 supernatant. The A4 cell line appeared to be a null cell in respect to the P-SC cell line properties; only the slight amount of immunoglobulin produced suggested that the A4 cell line is of the B cell lineage. An association of viral particles with cells of the A4 morphology and null antigenic characteristics was observed and may provide an explanation for the reduced B cell properties of A4 cells.

Cellular defense of the avian respiratory system: influx and nonopsonic phagocytosis by respiratory phagocytes activated by Pasteurella multocida.

Poultry have a very limited number of resident macrophages in the normal steady-state respiratory tract. Thus, poultry must rely heavily on active migration of phagocytic cells to the lungs and air sacs in defending against respiratory pathogens. Intratracheal administration of a live, apathogenic Pasteurella multocida vaccine (Choloral; Clemson University strain) increased the number of avian respiratory phagocytes (ARP; obtained by lavage of lungs and air sacs) within 24 h by 3 orders of magnitude compared with the number of ARP obtained from mock-inoculated controls and from nonreacting chickens. Chickens yielding a high number of ARP did not show any sign of respiratory disease. Flow cytometric analysis of ARP that were exposed to 20 nonopsonized fluorescent microspheres per ARP for 30 min at 37 degrees C demonstrated a fivefold increase in the percentage of actively phagocytic cells in the ARP populations of stimulated chickens compared with the percentage of phagocytic ARP for mock-inoculated control birds. The phagocytic capacity (relative number of engulfed microspheres) of ARP from stimulated birds doubled during the same time. The flow cytometric observations were confirmed by fluorescence microscopy. These results indicate that activation by avirulent replicating agents of phagocytic cells of chicken to migrate to the respiratory tract may be a means of defending poultry against air sacculitis and pneumonia.