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Spontaneous rosette formation and rosette inhibition assays in patients with squamous cell carcinoma of the head and neck.

Peripheral blood lymphocytes from patients with squamous cell carcinoma (SCC) of the head and neck were studied by spontaneous lymphocyte rosette and rosette inhibition (RI) assays prior to treatment. The patients were clinically staged and the results of the assays compared with the clinical stage of the disease. The percentage of T-lymphocytes as determined by the spontaneous lymphocyte rosette test was significantly lower (p less than .01) for the patient group when compared with a normal population. Patients with stage I and II disease did not differ significantly from controls. Individuals with stage III or IV disease, however, had significantly lower T-lymphocyte counts. The tumors were histologically graded as well, moderately well, or poorly differentiated SCC. Patients with poorly differentiated neoplasms had significantly lower T-cell counts. The RI assay (using horse anti-human thymocyte globulin to inhibit rosette formation) was abnormal in many of the patients but did not appear to be a more sensitive in vitro measure of cell mediated immunity in these patients. Performing both tests detected more patients with cellular immunologic incompetence than either one alone.

Adolescent

Rosette formation between human lymphocytes and sheep erythrocytes. Inhibition of rosette formation by specific glycopeptides.

Rosette formation with unsensitized sheep erythrocytes is a characteristic of human thymus dependent lymphocytes. Release of glycopeptides from the sheep erythrocyte by trypsin reduces rosette formation. These tryptic glycopeptides inhibit rosette formation by untrypsinized sheep erythrocytes; this suggests that rosetting is mediated by erythrocyte surface glycopeptides. To investigate the molecular nature of this interaction, we examined the abilities of various model compounds to act as haptenic inhibitors of rosette formation. Inhibition is given by glycopeptides bearing oligosaccharide units rich in sialic acid, galactose, N-acetylglucosamine, and mannose linked to asparagine residues through glycosylamine bonds. Among compounds tested, fetuin glycopeptide is most effective, but human transferrin glycopeptide and human erythrocyte glycopeptide I also inhibit rosette formation. Other compounds including human erythrocyte glycopeptide II, human IgG glycopeptide, lacto-N-neotetraose, 3'- and 6'-sialyllactose show no significant inhibition. Neither sialic acid, galactose, manose, nor N-acetyl-glucosamine alone inhibits rosette formation. Stepwise degradation of fetuin glycopeptide established the galactose residues as important determinants of inhibitory activity. Fetuin glycopeptide blocks rosette formation when added to a suspension of human lymphocytes and sheep erythrocytes or when preincubated with human lymphocytes, but not when preincubated with sheep erythrocytes. Studies of the binding of [3H] fetuin glycopeptide to normal lymphocytes demonstrate 7.5 x 10(6) saturable binding sites per cell. No saturable binding of this compound to sheep erythrocyte membranes is observed. Compared to normals, lymphocytes from patients with chronic lymphatic leukemia demonstrate decreased fetuin glycopeptide binding with a mean of 0.9 x 10(6) sites per cell. This decreased binding correlates with the impaired ability of these cells to form rosettes. The data suggest that fetuin glycopeptide inhibits rosette formation by binding to the thymus-dependent cell where competition occurs with sheep erythrocytes for specific lymphocyte surface receptors.

Animals

Abnormal spontaneous rosette formation and rosette inhibition in lung carcinoma.

Fifty-five children with CID and known ADA status were studies at a workshop held in Albany, New York. Erythrocyte ADA determinations were performed in 22 of the 55 patients, 13 of whom were ADA negative. The ADA defect appears to be transmitted as an autosomal recessive trait. Some patients with CID and ADA deficiency have characteristic radiologic abnormalities of the skeleton, which are not found in other illnesses. The thymus glands of all patients with CID and ADA deficiency who could be examined have evidence of thymic involution manifested by presence of Hassall's corpuscles and differentiated germinal epithelium; this is in contrast to "classic" thymus findings in CID with normal ADA. Adenosine deaminase probably plays an important, although as yet undefined, role in lymphocyte development and/or function. The deficiency of ADA in CID is the first enzyme defect observed in a deficiency disease of specific immunity.

Aged

Detection of nerve growth factor binding sites on neuroblastoma cells by rosette formation.

A cloned suspension culture of mouse C1300 neuroblastoma cells bound, at 2degrees, sheep erythrocytes passively coated with nerve growth factor, with the formation of rosettes. When grown in tissue culture dishes to which they could attach, neuroblastoma cells rapidly transformed, within 48 hr emitting cytoplasmic processes some of which were several mm long. Most of the attached neuroblastoma cells formed rosettes. In contrast, normal mouse kidney cells or various murine tumor cell lines used as cell controls exhibited a poor capacity for binding nerve growth factor. Rosette formation was a specific reaction that could be prevented by pretreating cells with proteolytic enzymes, free nerve growth factor, or specific antibodies against neuroblastoma cell extracts.

Animals

[Participation of antibodies synthesized in vitro in rosette formation].

The author studied a possibility of rosette-formation by cells which became bound with the antibodies synthesized in vitro. It was found that antibodies capable of inducing formation of false rosettes appeared at the period of conduction of the rosette-formation reaction at 37 degrees C and with the prolongation of the incubation time in the culture medium. In carrying out the reaction of rosett-formation under conditions of cold the formation of false rosettes in the precipitate after the centrifugation was minimal.

Animals

Modulation of human T lymphocyte rosette formation by autonomic agonists and cyclic nucleotides.

Early rosette formation by T lymphocytes appears to be modulated by cyclic nucleotides. Dibutyryl cyclic 3',5' adenosine monophosphate (cyclic AMP) 10(-2) M inhibited E rosette formation up to 83%, while 10(-6) M 8-bromo-cyclic guanosine monophosphate (cyclic GMP) increased rosette formation maximally to 67.4% with less pronounced effect at increased concentrations. T lymphocyte receptors for beta adrenergic, alpha adrenergic, and muscarinic cholinergic agonists appear to influence rosette formation. Isoproternol 10(-2) M induced 67.3% inhibition, while phenylephrine 10(-5) M and carbamylcholine 10(-4) M were associated with enhanced rosette formation of 67.2% and 57.8%, respectively. Selective blockade with propranolol, phentolamine, and atropine indicated the presence of separate receptor sites. The lack of effect of histamine at concentrations of 10(-3) M and above suggests the absence of specific recepotrs on peripheral human T lymphocytes.

Atropine

The effect of varying SRBC/lymphocyte ratio on T cell rosette formation.

To determine the effect of varying the SRBC/lymphocyte ratio on rosette formation, rosettes were formed at SRBC/lymphocyte ratios ranging from 10 to 200. To assess the significance of the time of incubation, duplicate samples were mixed at various SRBC to lymphocyte ratios and the percentage of rosette-forming lymphocytes was determined immediately or after an 1-hour incubation at 4 degrees C. The results of five experiments revealed the highly statistically significant difference between the percentage of rosette-forming cells in tests performed with (higher number of RFC) and without (lower number of RFC) incubation, in all SRBC/lymphocyte ratios. On the other hand, ratio of SRBC to lymphocytes influenced significantly the results obtained in both types of tests, this effect was more pronounced in test performed with 1-hour incubation. Optimal SRBC/lymphocyte ratios for both tests were 100:1 (31% RFC without incubation and 53.4% of RFC after 1-hour incubation.

Animals

The significance of varying SRBC/lymphocyte ratio in T cell rosette formation.

The incubation ratio of sheep red blood cells (SRBC) to lymphocytes is a critical factor in rosette formation, whereas the length of time SRBC and lymphocytes are incubated together does not significantly affect the percentage of lymphocytes forming rosettes. The graph obtained by plotting percentage of rosette formation against the ratio of SRBC to lymphocytes is similar to that resulting from the formation of bimolecular complexes. If rosette formation is analogous to formation of bimolecular complexes, maximal rosette formation occurs when the system is saturated, i.e., with excess SRBC, and is a measure of the total capacity of a lymphocyte population to form rosettes. In addition, the percentage of rosette formation observed at a limiting SRBC/lymphocyte ratio gives an indication of the avidity of the lymphocytes for SRBC. This interpretation may provide an explanation for the difference between the "active" and "total" rosettes. When the log of the SRBC/lymphocyte ratio is plotted against percentage of rosette formation, a straight line is obtained, suggesting that within a given normal lymphocyte sample, T cell subsets with different avidities are not detected by rosette formation at different SRBC/lymphocyte ratios.

Blood Cell Count

Rosette formation assays in dogs: lack of specificity of E rosettes for T lymphocytes.

The present study examined the specificity of guinea pig erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosette formation assays with suspensions of canine peripheral blood lymphocytes. Neutrophils, monocytes, and lymphocytes bound EAC but not erythrocyte-antibody (EA) controls. Similarly, all three cell types formed rosettes with guinea pig E. Adherence of guinea pig E to these cells was apparently mediated by natural cytophilic antibodies present in the serum used in the suspension medium. The nonspecificity of the guinea pig E-rosette formation assay with canine lymphocytes renders the technique unreliable for the identification of thymus-derived lymphocytes in dogs.

Animals

In vitro radiosensitivity of human T and B lymphocytes evaluated using lymphocyte transformation tests and rosette formation tests.

The influence of irradiation upon human lymphocytes was studied using lymphocyte transformation tests and formation of E and HEAC rosettes. Irradiation was given in vitro using doses between 0 and 50,000 rad. It was shown that blast transformation after stimulation with T-cell stimulating agents (as PHA, PWM, Con.A and PPD) was suppressed by irradiation. The effect of irradiation upon T lymphocytes was also shown in different kinds of MLC experiments. Both the effect of irradiation upon rosetts formation and the influence of irradiation upon already formed rosettes were analyzed. The ability of lymphocytes to form E rosettes was affected after irradiation with 500 rad: there were fewer E rosettes with 3 SRBCs, decrease in total number of E rosettes and more null cells, with no depression of the number of HEAC rosettes formed. Already formed E and HEAC rosettes were totally unaffected of irradiation, and this radioresistance was also observed for 18-hour-old rosettes. The ability to form spontaneous E rosettes was decreased after irradiation of the lymphocytes with 100 rad; increasing doses did not cause further depression, and already formed spontaneous E rosettes were radioresistant. The mechanisms involved in E-rosette formation thus seem to be radiosensitive.

B-Lymphocytes

A comparative study of procedures for sheep erythrocyte-human-T-lymphocyte rosette formation.

A comparative study of several published methods for sheep red blood cell-T-lymphocyte rosette formation was performed. Maximum SRBC-rosette formation occurred with AET treated SRBC in medium supplemented with 20% FCS or with untreated SRBC in 100% FCS. Prolongation of the 4 degrees C incubation period from 4 to 18 h enchanced rosette formation. Fluorescein diacetate staining significantly increased calculated percentage of rosette formation. Fluorescein diacetate staining significantly increased calculated percentage of rosette-forming lyphocytes by allowing accurate indentification of the central lymphocytes in morulas.

Animals

Lack of effect of halothane on E, EA, and EAC rosette formation and cyclic AMP level of human lymphocytes.

The effect of halothane on rosette formation and cyclic adenosine monophosphate (cyclic AMP) level of human lymphocytes was investigated. A 4- or 24-hour incubation with 2% halothane had no significant effect on "early" or "late" E, EA and EAC rosette formation or intracellular cyclic AMP levels of human lymphocytes whereas cytochalasin B, which affects microfilament action, reversibly inhibited both E and EA but not EAC rosette formation. It is suggested that halothane in clinical concentrations neither disturbs the function of microfilaments, essential for E and EA rosette formation, nor affects the metabolism of cyclic AMP in unstimulated human lymphocytes.

Cells, Cultured

Restoration by levamisole of histamine-inhibited E rosette formation of T lymphocytes of patients with allergies.

Histamine significantly inhibited the E rosette formation of T lymphocytes of patients with allergies in vitro, but had no effect on the E rosette formation of T lymphocytes of healthy subjects. This inhibitory effect was obtained by concentrations of 3.10(-4) down to 3.10(-7) M histamine and depended on the time of incubation. Levamisole an antianergic chemotherapeutic agent, could restore the histamine-inhibited E rosette formation. It is postulated that patients with allergies possess a subpopulation of T lymphocytes bearing histamine receptors and that the inhibitory effect of histamine on E rosette formation is medicated by intracellular cAMP.

Adolescent

Inhibition of rosette formation of IgE-bearing rat mast cells by disodium chromoglycate.

The interaction of reaginic antibody with specific antigen was studied by the rosette formation of peritoneal rat mast cells. The mast cells were obtained from actively sensitized rats or were passively sensitized in vitro. Rosette formation was of a higher degree with mast cells of actively sensitized rats; in this case 58% of the cells showed a strong rosette-forming effect (blinding more than 5 SRBC). No rosette formation was detected in 18% of the cells. With passively sensitized rat mast cells, rosette formation was 45% and 22%, respectively. Rosette formation of both actively and passively sensitized mast cells could be inhibited by disodium chromoglycate (DSCG); the inhibitory effect of 20 micrograms and 200 micrograms of the drug was the same, and neither dose caused a full inhibition. It is suggested that the linkage of specific antigen to the surface of sensitized mast cells can be inhibited by DSCG in vivo.

Animals

Rosette formation by canine peripheral blood lymphocytes.

The surface membrane characteristics of canine peripheral blood lymphocytes were investigated using erythrocyte (E) and erythrocyte antibody complement (EAC) rosette assays and immunofluorescent staining techniques. Canine thymus-derived lymphocytes formed nonimmune E rosettes with human and guinea pig erythrocytes at approximately the same percentages (mean percentage 36.3 and 32.4, respectively). Rosettes did not form with erythrocytes from seven other animal species. Cell surface immunoglobulins were demonstrable in a small percentage (4%) of the E rosette-forming lymphocyte population. Fragments of human erythrocytes inhibited E rosette formation by intact human red cells, but did not result in a significant decrease in rosette formation by intact guinea pig erythrocytes; likewise, guinea pig fragments had no inhibitory effect on rosette formation by human erythrocytes, demonstrating that separate receptors were required for the two red cell types. EAC rosette formation was not affected by addition of intact or fragmented human and guinea pig erythrocytes. Canine bone marrow-derived lymphocytes were characterized by immunoglobulin on the cell surface and EAC rosette formation. Serial tests of lymphocytes from one dog revealed a wide variation in percentage of cells forming E and EAC rosettes. A close correlation was observed between the immunoglobulin-bearing cells (mean percentage 46.6) and those forming EAC rosettes (mean percentage 49.3).

Animals

Nonimmune rosette formation: a measure of the newborn infant's cellular immune response.

Nonimmune rosette formation, an in vitro measure of cellular immunity, was evaluated in normal newborn infants. Active rosette formation in 14 specimens of cord blood was 18.9 +/- 4.8% compared to 28.1 +/- 5.2% in 15 adult control samples (p less than 0.05). Total rosette formation in 13 cord blood samples was 33.3 +/- 7.6% compared to 55.1 +/- 6.5% in 15 adult control specimens (p less than 0.05). Tritiated thymidine uptake from phytohemagglutinin stimulation was comparable in cord blood and adult control lymphocytes. The importance of these findings is discussed in light of other recent reports suggesting that cord blood thymic derived T lymphocytes may have reduced immune capability when compared to adult lymphocytes.

Fetal Blood

Human lymphocyte subpopulations: rosette formation with sheep, human and horse red blood cells.

Rosette formation between human lymphocytes and horse red blood cells could be promoted by a low pH medium, overnight incubation and a temperature of 4 degrees C. The percent of sheep, horse and human rosette-forming cells in the peripheral blood were 71.7 +/- 1.8, 30.5 +/- 2.8 and 28.3 +/- 3.4 respectively. However, their percentages in thymuses were 97.1 +/- 1.1, 91.4 +/- 2.4 and 89.0 +/- 3.4. Using preparations of isolated subpopulations, it was observed that the horse and human red cell rosette-forming cells were probably also "early" sheep red cell rosette-forming cells. Rosette formation with all three types of red blood cells were inhibited by a preparation of Fetuin-glycopeptide.

Animals

RRBC and EAC rosette formation after DNCB contact sensitization and cyclophosphamide treatment of guinea-pigs.

Rabbit erythrocyte (RRBC) and EAC rosette formation, for detection of T and B lymphocytes respectively, was studied in thymus, lymph nodes and peripheral blood of guinea-pigs. Using two different methods for preparation of RRBC rosette-forming cells (RFC) it was found that, without an incubation period at 37 degrees, a smaller part of the pool of T cells is detected which is particularly sensitive to cyclophosphamide (cy). RRBC rosette formation was studied for 2 weeks after treatment with one high dose of cy and appeared to be minimal in the thymus after 1 week and maximal in the lymph nodes at about day 3. EAC rosette formation was completely blocked by cy treatment. Four days after contact sensitization with 2,4-dinitrochlorobenzene (DNCB) no percentual increase was found in RRBC-RFC, suggesting a parallel increase of B and T cells in stimulated lymph nodes. Cytological screening of the rosettes at day 4 after DNCB showed that of the RRBC-RFC approximately 17 per cent were blast cells, while no difference was detected in the number of blast cells participating in EAC rosette formation as compared to the unstimulated lymph nodes. In contrast to RRBC-RFC the percentage of EAC-RFC in draining lymph nodes is increased after contact sensitization. The mechanism of this increase is discussed.

Animals