Rudolph S. Parrish

Phenotypic and functional reconstitution of peripheral blood lymphocytes following T cell-depleted bone marrow transplantation from partially mismatched related donors

Myeloablative chemotherapy followed by transplantation of a T cell-depleted bone marrow graft from a partially mismatched related donor provides a potentially curative option for patients with leukemia and other disorders of hematopoiesis, although the patient is faced with a period of sustained immunodeficiency as well as pharmacologic immunosuppression as a result of prophylaxis against graft-versus-host disease. Thirty patients who received one to three antigen T cell-depleted mismatched grafts were evaluated for immune reconstitution. The percentage and numbers of cells expressing lymphocyte subset antigens were determined by flow cytometry at 14, 28, 60, 100, 180, 270 and 365 days post-BMT and at 6 month intervals thereafter. Lymphocyte reconstitution was characterized by the early appearance of natural killer cells and a low percentage of both T and B cells. During the first year after BMT, the number of NK cells remained constant while T and B cells gradually returned to normal numbers and proportions. Response to the lymphocyte mitogen phytohemagglutinin returned to normal over the course of 2 years, while the response to concanavalin A was slightly depressed and the response to pokeweed mitogen became supranormal at about 1.5 years and continued to increase. These data suggest the need for long-term immunophenotypic monitoring as well as prolonged infection surveillance and prophylaxis.

Influence of T cell depletion method on circulating γδ T cell reconstitution and potential role in the graft-versus-leukemia effect

Background Our laboratory previously reported that leukemia patients who developed ≥ 10% γδ + T cells during the first six months after receiving an anti-TCRαβ T-cell-depleted (TCD) graft from a partially mismatched related donor (PMRD) had a disease-free survival (DFS) advantage. These γδ + T cells were Vδ1 + CD3 + CD4 − CD8 − CD69 + HLADR + and are cytotoxic to K562 cells. Methods In order to determine whether the anti-αβ TCD regimen was associated with these findings, we compared the reconstitution of γδ + T cells from patients who received TCD PMRD grafts using the anti-TCRαβ MAb T10B9-1A31 (previously reported) with similar patients who received grafts using the anti-CD3 MAb OKT3. Results Increased cytotoxic Vδ1 + T cells were seen in 10 of 43 T10B9 TCD patients compared to 7 of 100 in the OKT3 TCD group (23% versus 7%, p=0.010). T10B9 patients with increased γδ + T cells also exhibited a higher range of increased γδ + T cells and the length of time the γδ + T cells remained high was longer when compared to OKT3 patients. Patients with increased γδ + T cells whose grafts were T-cell depleted with T10B9 showed a significant decrease in relapse (p = 0.038). Similar rates and reduction in relapse were seen in OKT3 TCD patients, although significance was not reached due to the small number of patients with increased γδ + T cells. Estimated 3 year disease-free survival was significantly improved in T10B9 patients with increased γδ + T cells (0.79 versus 0.31, p=0.009), a trend also seen in OKT3 patients (p = 0.091). Discussion These observations indicate that Vδ1 + CD4 − CD8 − cytotoxic T cells are associated with lower relapse rates and improved survival, and thus may have a role in a graft-versus-leukemia effect.

Expression of CD134 (0X-40) on T cells during the first 100 days following allogeneic bone marrow transplantation as a marker for lymphocyte activation and therapy-resistant graft-versus-host disease.

CD134 (OX-40) is an activation-associated antigen which functions as a costimulatory receptor for CD4+ T cells. In order to determine the expression of CD134 during immune recovery following allogeneic bone marrow transplantation (BMT), we measured its expression on T cells and T cell subsets during the first 100 days following BMT in 26 patients. CD4+CD134+ T could be seen approximately 14 days following BMT cells in patients who did not develop GvHD which required therapy (n = 20). The percentage of CD4+CD134+ cells continued to increase up to the fourth week following BMT to a maximum of 40-50% of CD4+ T cells (normal = 1-8%). Two patients who developed Grade I-II GvHD and who responded to treatment with pulsed high-dose methylprednisolone (MPD) showed a decline of approximately 40% in CD4+CD134+ T cells was seen within 48 hours of treatment. Four patients who developed GvHD that was not responsive to MPD and who later developed high IV GvHD showed increasing CD4+CD134+ T cells up to 85% of the CD4+ T cells. Additionally, rapid increases in CD134+ T cells following antibody-based T cell therapy were associated with GvHD recurrence. In no cases was the percentage of CD134+ CD4+ T cells predictive of clinical GvHD. In this exploratory study, we have shown that CD134, although not predictive of the initial onset of GvHD, may be a useful tool for monitoring the response to early GvHD therapy and identification of patients at risk for reemergence of GvHD who may benefit from anti-T cell therapy. Cytometry (Comm. Clin. Cytometry) 38: 238-243, 1999.

Within-person variability of the ratios of urinary 2-hydroxyestrone to 16α-hydroxyestrone in Caucasian women☆ ☆

The ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) has been suggested as a potential biomarker for breast cancer risk. We evaluated within-person variability of this biomarker in ten healthy Caucasian women aged 23-58 years. Each study participant was asked to provide an overnight fasting morning urine sample once a week for an average of 8 weeks. These urine samples were assayed for 2-OHE1 and 16alpha-OHE1 by using competitive enzyme immunoassay kits purchased from the ImmunaCare Corporation. The coefficients of variation for urinary 2-OHE1/16alpha-OHE1 over the study period ranged from 13.7 to 59.6% (mean, 33.3%) in our study participants. There was a good correlation between the level of the urinary 2-OHE1/16alpha-OHE1 ratio in any single urine sample and the average ratio over the 8-week study period from the same woman, with the mean correlation coefficient of 0.85. These results indicated that the within-person variation of the 2-OHE1 to 16alpha-OHE1 ratio for most women was moderate and the level of this ratio in a single urine sample, in general, reflects reasonably well the level of this biomarker over a 2-month period.

A field study to compare performance of stainless steel research monitoring wells with existing on-farm drinking water wells in measuring pesticide and nitrate concentrations

Existing drinking water wells are widely used for the collection of ground water samples to evaluate chemical contamination. A well comparison study was conducted to compare pesticide and nitrate-N data from specially designed stainless steel research monitoring wells with data from nearby existing on-farm drinking water wells. Results could help to determine whether adequate information concerning ground water contamination can be obtained from existing drinking water wells for use in making pollutant control decisions. The study was conducted during 1993-1994 in the Little Coharie Watershed, a 158 square mile area located in the coastal plain of eastern North Carolina. Statistical analysis indicated that research monitoring wells provided a greater probability of detecting pesticides in ground water than existing on-farm wells. Atrazine was the most frequently detected pesticide found in all wells, followed in order by fluometuron, carbofuran, metolachlor, alachlor, carbaryl, butylate, chlorothalonil, linuron and simazine. Ninety-seven percent of all wells had observed concentrations of nitrate-N, ranging from 0.1 to 30.1 mg/L. There was not a significant difference between research wells and existing wells for monitoring nitrate-N. Based on results of this study, existing drinking water wells can be used for monitoring nitrate; however, specialized stainless steel monitoring wells should be used for monitoring pesticides in ground water.

A Multivariate Approach for Assessing Severity of Acute Graft-Versus-Host Disease in Bone Marrow Transplantation

Patients undergoing bone marrow transplantation are at high risk of developing acute graft-versus-host disease (GVHD) which is a primary limiting factor for this procedure inasmuch as it is responsible for high morbidity rates and is associated with poor survival outcome. To provide improved treatment assessment and better interpretation of clinical outcomes, we need a precise and objective assessment of GVHD. Severity of GVHD is commonly assessed using an imprecise categorical grading system that incorporates skin, gut and liver grades, as well as subjective assessment of clinical performance. These organ grades are based on arbitrary cutpoints of skin rash, diarrhoea volume and bilirubin level. The International Bone Marrow Transplant Registry proposed an alternative grading system based on different combinations of organ involvement and provided estimates of relative risk of treatment failure. On the basis of that work, we developed an empirical mathematical model that quantifies GVHD severity, and that uses continuous, rather than categorical, daily measurements for each organ system. We use model-predicted values as an index of severity for any combination of values. The proposed index allows a more precise comparison of GVHD profiles across different treatment protocols and also permits more refined analyses to address relationships between GVHD and clinical outcomes.

DESIGN AND ESTIMATION IN THE LIMITING DILUTION ASSAY WHEN CONCENTRATIONS VARY WIDELY AMONG SAMPLES

Limiting dilution assays (LDA) are used to estimate an unknown cell fraction of interest within a sample. This paper discusses a method for designing an LDA using the distribution of the cell fraction of interest, examining three different design approaches: geometric progression, equally spaced log, and equiprobability. Two common estimation methods, minimum chi-square and maximum likelihood, also are investigated. These designs and estimation methods, coupled with varying numbers of wells per dilution and dilutions per design, are compared quantitatively through computer simulation. Performance measures computed were mean relative bias and mean squared error.