Tannic acid-stained microtubules with 12, 13, and 15 protofilaments.

Subunit structure in the walls of sectioned microtubules was first noted by Ledbetter and Porter (6), who clearly showed that certain microtubules of plant meristematic cells have 13 wall protofilaments when seen in cross section. Earlier, protofilaments of microtubular elements had been described in negatively stained material, although exact counts of their number were difficult to obtain. In microtubular elements of axonemes, some success has been achieved in visualizing protofilaments in conventionally fixed and sectioned material (8, 10); much less success has been achieved in identifying and counting protofilaments of singlet cytoplasmic microtubules. By using glutaraldehyde-tannic acid fixation, as described by Misuhira and Futaesaku (7), Tilney et al. (12) studied microtubules from a number of sources and found that all have 13 protofilaments comprising their walls. These authors note that "...the number of subunits and their arrangement as protofilaments appear universal...". Preliminary studies of ventral nerve cord of crayfish fixed in glutaraldehyde-tannic acid indicated that axonal microtubules in this material possess only 12 protofilaments (4). On the basis of this observation, tannic acid preparations of several other neuronal and non-neuronal systems were examined. Protofilaments in microtubules from these several cell types are clearly demonstrated, and counts have been made which show that some kinds of microtubules have more or fewer protofilaments than the usual 13 and that at least one kind of microtubule has an even rather than an odd number.

Alterations in number of protofilaments in microtubules assembled in vitro.

Tubulin from bovine brain was polymerized in vitro using a variety of assembly conditions. Many of the formed microtubules were shown to contain 14 wall protofilaments. The number of microtubules containing 14 protofilaments increased with consecutive repetitions of cold-dissociation followed by reassembly in vitro.

Unrelated donor or partially matched related donor peripheral stem cell transplant with CD34+ selection and CD3+ addback for pediatric patients with leukemias.

Unmodified peripheral stem cell transplants are associated with an increased risk of extensive chronic GVHD. T depletion may reduce this risk, but the risk of graft failure or relapse may increase. To decrease the risks of both extensive chronic GVHD and graft failure, we added back a defined dose of CD3+ cells to CD34+ selected PSCs. Twenty-four patients were evaluable for outcome analysis. Donors were unrelated (23) or related (1). Conditioning was thiotepa, cyclophosphamide, and total body irradiation. Cyclosporine was used post transplant. Following CD34+ selection, a total of 5 x 10(5)/kg CD3+ cells were infused. Donors were matched for 12 patients. The median CD34+ dose infused was 7.1 x 10(6)/kg. Engraftment occurred in all patients at a median of 14 days (10-19). Twelve patients are alive in remission 15-34 months (median, 25) post PSCT. GVHD occurred in 17 patients, but was >grade II in only 2. Chronic GVHD occurred in 61.5% of evaluable patients, but was limited to skin and perioral cavity. Two patients relapsed, and 10 patients died of non-relapse causes. This study demonstrates that PSCT with CD34+ selection and a defined dose of CD3+ results in prompt engraftment and may limit development of extensive chronic GVHD.

Unrelated donor bone marrow transplantation for children with severe aplastic anemia: minimal GVHD and durable engraftment with partial T cell depletion.

Both increased graft rejection and increased graft vs host disease (GVHD) remain obstacles to success for unrelated donor (URD) BMT for patients with SAA. Partial T cell depletion (PTCD) may decrease the risk of severe GVHD, while still maintaining sufficient donor T lymphocytes to ensure engraftment. We report on 12 patients with SAA who underwent PTCD URD BMT. All patients had failed medical therapy or relapsed following initial responses, and were transfusion dependent. The median age was 6 years, and there were five males. Donors were matched for four patients, and mismatched for eight. All patients received total body irradiation with either Ara-C or thiotepa and cyclophosphamide. PTCD was accomplished using monoclonal antibody T10B9 or OKT3 and complement. All patients engrafted, with a median time of 18 days to ANC >500. Only one patient had greater than grade II acute GVHD; two patients had limited and one patient extensive chronic GVHD. Nine patients are alive and transfusion independent at a median months post BMT. Three patients died from infection or renal failure. This series suggests that an aggressive immunosuppressive conditioning regimen with PTCD results in successful engraftment and minimal GVHD in pediatric patients with SAA, even with HLA mismatched donors.

Outcomes of transplantation with partial T-cell depletion of matched or mismatched unrelated or partially matched related donor bone marrow in children and adolescents with leukemias.

Graft-versus-host disease (GVHD) remains a major barrier to successful hematopoietic stem cell transplant for patients who lack a matched related donor. Partial T-cell depletion (TCD) of the graft may decrease the risk of severe GVHD with unrelated donors (URD) and partially matched related donors (PMRD) while retaining an antileukemic effect. We analyzed our experience using URD and PMRD for pediatric patients with leukemias from 1990 to 2001. A subgroup of 'matched' URD donor pairs was retrospectively analyzed for high-resolution class I. Partial TCD was accomplished with monoclonal antibody T10B9 or OKT3 and complement. There were 76 URD (45% matched) and 28 PMRD recipients. Event-free survival (EFS) was 38.3%, and overall survival (OS) 45.1% at 3 years. On multivariate analysis, there was no difference in survival based upon marrow source, but nonrelapse mortality was higher with the use of PMRD. Relapse occurred in 6% of ALL patients, and 22.8% of AML/MDS patients. Grades III-IV GVHD was observed in only 6.7% of patients. Partial TCD allows use of matched or mismatched URD, or PMRD with little mortality from GVHD, durable engraftment, and no increase in relapse risk.

Angiogenesis inhibitor TNP-470 during bone marrow transplant: safety in a preclinical model.

High-dose therapy with stem cell rescue is a treatment option for patients with advanced solid tumors. Although this approach has promise for some pediatric cancers, especially neuroblastoma, it is limited by the risk of relapse posttransplant as well as concern about possible reinfused tumor cells in autologous stem cell products. Antiangiogenic agents given during and after recovery from high-dose therapy with stem cell rescue may decrease the risk of relapse. TNP-470 is an antiangiogenic agent now in clinical trials. Although it inhibits the growth of bone marrow (BM) colony-forming cells in vitro, no significant hematological toxicity has been seen in Phase I trials. To assess the feasibility of using antiangiogenic agents during the period of posttransplant hematopoietic engraftment, we have developed a model of stem cell transplant in mice. Mice were lethally irradiated and then rescued with stem cells containing a transgene expressed in the hematopoietic lineage. Mice were then treated with TNP-470 or placebo, and assessed for survival, successful engraftment, and kinetics of engraftment. Both treated and control mice demonstrated reliable multilineage engraftment as well as normal lymphoid maturation with no excess mortality in the treated group. WBCs were lower but still within the normal range at d+28 in mice treated with bolus TNP-470, but not in those treated with continuous infusion TNP-470, compared with controls. These data indicate that inhibitors of angiogenesis do not adversely impact engraftment after stem cell transplantation.

The rat testicular A1 adenosine receptor-adenylate cyclase system.

When adenosine interacts with membrane-bound A1 receptors, it is capable of inhibiting the enzyme adenylate cyclase in brain and fat tissue. In this paper we characterize the A1 adenosine receptor-adenylate cyclase system of the rat testes. The agonist radioligand (-)-N-[3-[125I]iodo-4-hydroxyphenyl-(isopropyl)adenosine binds with high affinity (Kd, congruent to 1 nM) in a saturable manner (maximum binding, congruent to 600 fmol/mg protein). The A1 adenosine receptor binding displays the appropriate pharmacology, stereospecificity, and sensitivity to guanine nucleotides. The testicular A1 receptor is also coupled in an inhibitory manner to the enzyme adenylate cyclase, as demonstrated by the ability of N6-R-phenyl-2-propyladenosine to inhibit isoproterenol- and forskolin-stimulated cAMP accumulation. The testicular A1 receptor can be solubilized in high yield and in an active form with the detergent digitonin. The solubilized receptor retains all of the pharmacological properties of the membrane-bound receptor. Although there are many similarities among the A1 receptor from testes, brain, and fat tissue, the testicular A1 receptor displays a larger apparent mol wt. By photoaffinity labeling, the A1 adenosine receptor-binding subunit of fat and brain are 38,000 mol wt proteins, while the testicular A1 receptor binding subunit is a 42,000 mol wt protein.

Susceptibility of human neuroblastoma cell lines to cytotoxic T lymphocyte-mediated lysis.

The susceptibility of neuroblastoma cells to cytotoxic T lymphocyte (CTL)-mediated killing was investigated. Cytotoxic lines were generated by sensitizing peripheral blood lymphocytes against two stimulator cells, a neuroblastoma line, CHP-100, and normal allogeneic lymphocytes, LS. LS cells shared class I antigens with CHP-100, but in addition expressed class II antigens. The resulting cell lines strongly lysed both CHP-100 and LS cells, but poorly killed the natural killer (NK) target K562. Specific blocking of lysis by a monoclonal antibody directed against class I determinants and strong killing by the line following depletion of cells with NK or LAK markers demonstrated that this neuroblastoma line was lysed by CTL.

The use of cytotoxic T cell lines to detect the segregation of a human minor alloantigen within families.

A cytotoxic T cell (CTL) line, which detected a minor alloantigen provisionally called W was generated in vitro with lymphocytes from a multiply transfused individual, S1. Lymphocytes from S1 were first stimulated with cells from an unrelated known from previous studies to express the minor antigen. The primary CTL were then restimulated with cells from a W +/ve HLA identical sib, S2, in the presence of IL-2. As in previous work, recognition of the W antigen by these CTL was restricted by HLA-B7. Antigen assignments of W + W -, based upon cold target inhibition studies, confirmed previous assignments which had depended upon the ability of lymphocytes either to stimulate the generation of or to be killed by anti-W CTL effectors. Testing of lymphocyte targets from members of several unrelated families in which HLA-B7 segregated showed that the CTL lines could detect the expression of W on cells of individuals in the general population. In 3 of 5 cases, members of an HLA identical sib pair differed for W. These results open up the possibility of designing studies using CTL lines to determine whether differences for minor alloantigens play a role in clinical transplantation.

Study of a human minor alloantigen in relation to clinical graft-versus-host disease.

Sixty-seven pairs of HLA matched siblings, each comprising marrow donor and recipient in the Seattle marrow transplant program, were analyzed to establish phenotype for a newly described minor H antigen, W1. The test for phenotype entailed cold target inhibition of cytotoxicity, directed at this antigen and mediated by specifically stimulated T cell lines. There were 58 compatible and six W1 incompatible pairs. The low frequency of W1 mismatch is due to the strong preponderance of W1-positive individuals in the general population. Severe graft-versus-host disease (GVHD), both acute and chronic, was observed among the 58 recipients of marrow from W1 matched donors. These results do not reveal any particular importance for W1 incompatibility in human GVHD and indeed indicate that other systems are involved. Even if some cases are triggered by incompatibility at W1, the maximum frequency with which this could occur would be about 10%, due to the limited polymorphism of this alloantigenic system.

Wall substructure of microtubules polymerized in vitro from tubulin of crayfish nerve cord and fixed with tannic acid.

Microtubules of axons of crayfish nerve cord normally have 12 wall protofilaments and microtubules of surrounding glial cells have 13 protofilaments. Tubulin was isolated from such nerve cords and polymerized in vitro; tannic acid staining of sedimented microtubules showed that microtubules with 12 and 13 wall protofilaments were present, suggesting that the ability to form a microtubule with a given number of protofilaments is inherent in the tubulin or its associated proteins. The temperature of polymerization was found to influence the number of protofilaments in resultant microtubules. With assembly at 20 degrees C, for example, most of the complete microtubules had 13 protofilaments, while at 40 degrees C most showed 10 protofilaments. It is suggested that the temperature of in vitro polymerization, among other factors, can influence the angle of binding between adjacent protofilaments and thus alter the number of protofilaments required to complete the circumference of the tubule.

Cytotoxic T lymphocyte lines (CTLL) against a human minor alloantigen.

Cytotoxic T-cell lines were generated following in vitro culture of lymphocytes from a patient suffering from aplastic anemia together with those of his HLA-identical brother, a repeated transfusion donor. The segregation pattern within the family of the determinant(s) detected by these cytotoxic cells strongly suggested that a minor alloantigen(s) was being detected. Testing of the effectors on a panel of unrelated individuals indicated that it was best seen in association with HLA-B7, which was common to both the patient and his sibling donor.

The development of cell mediated immunity: very young children have strong LAK activity.

We were interested in evaluating immune function in very young children with cancer who were treated with gamma-interferon on a sequential basis. Though gamma-interferon was reportedly able to enhance NK activity, and while many tumor cells are susceptible to lysis by these cells, this effector mechanism is not fully developed in very young children. Since LAK cells also have anti-tumor activity and are produced in response to stimulation with Interleukin-2, we investigated whether LAK killing might be more readily demonstrable in very young children. We report that LAK activity in this group did not differ significantly from that of adults. This was also true for a small group of neuroblastoma patients tested. Furthermore, as opposed to NK activity, LAK activity was demonstrable following freezing and thawing of PBL.