Mary Jane Hicks

The effects of 13‐cis‐retinoic acid and beta‐carotene on cellular immunity in humans

Deficiency of vitamin A and/or its precursors has been associated with increased cancer risk in animals and humans. Therapeutic trials of vitamin A and related compounds have demonstrated activity in several cancerous and precancerous conditions. The authors measured the effects of a retinoid, 13‐cis‐retinoic acid, and a carotenoid, beta‐carotene, on the human immune system in vivo in conjunction with their use in ongoing clinical trials. Immune cell subpopulations were analyzed by quantifying the expression of markers using flow cytometric study. Both compounds produced significant effects on immune cell populations. 13‐cis‐Retinoic acid resulted in an increase in the percentage of peripheral blood lymphoid cells expressing surface markers for T‐helper cells with only minimal effect on natural killer cell marker expression. In contrast, beta‐carotene produced an increase in the percentage of cells expressing natural killer cell markers with smaller effect on T‐helper markers. Modest increases in the percentage of cells expressing Ia antigen, transferrin, and interleukin‐2 receptors were produced by both drugs. These results suggest that retinoids and carotenoids can produce major changes in immune cellular marker expression in vivo in humans at doses relevant to their potential clinical use.

Immune function in chronic active Epstein-Barr virus infection

The spectrum of illness attributed to Epstein-Barr virus (EBV) includes patients with symptoms persisting for more than 1 year without any other obvious underlying disease. High titers of antibodies to EBV, either IgG anti-viral capsid antigen or anti-early antigen, can be demonstrated. In this study, 13 patients diagnosed as having chronic active EBV infection were examined to determine aspects of their immunologic status. Morphological examination and fluorescent antibody analysis revealed no abnormalities in the phenotypes of peripheral blood white cells present in these patients. Compared to those from healthy control individuals, mononuclear cells from the patients showed a markedly depressed ability to produce both interleukin-2 and interferon after stimulation with mitogen and a phorbol ester. Studies of natural killer (NK) cell activity revealed that unfractionated mononuclear cells from patients with chronic active EBV infection were significantly lower in killing activity compared to the control group. Fractionation procedures to enrich for large granular lymphocytes resulted in an increase in NK activity for all individuals, but killing activity still remained slightly lower in the patients than in the control group. The dysfunctions which were found in patients with chronic active EBV infection may reflect a primary defect in natural immune functions of the patients predisposing them to a chronic or intermittent clinical disease rather than a self-limiting illness. Alternatively, the abnormalities detected in these experiments may be a result of the viral infection itself.

Enhancement of the Expression of Activation Markers on Human Peripheral Blood Mononuclear Cells by In Vitro Culture With Retinoids and Carotenoids

Retinoids (retinol, retinal, retinoic acid, retinyl beta‐glucuronide, and 13‐cis retinoic acid) and carotenoids (beta‐carotene and canthaxanthin) were evaluated for their immunomodulatory effects on human peripheral blood T‐lymphocyte subpopulations and natural killer (NK) cells. Peripheral blood mononuclear cells (PBMC) from healthy young volunteers were isolated and incubated for 72 hours at various levels of retinoids and carotenoids including a physiological concentration (10‐8 M). Expression of surface antigens for total T cells, T‐helper and T‐suppressor cells, and activation markers (transferrin receptor, HLA‐Dr antigen, and interleukin 2 receptor) were analyzed with an EPICS V flow cytometer. Retinoic acid and 13‐cis retinoic acid (13‐cRA) produced significant increases in the percentage of cells with markers for total T‐helper cells, with a minimal effect on percentage of lymphocytes with markers for NK cells. However, beta‐carotene (BC), canthaxanthin (CTX), and retinyl beta‐glucuronide (RBG) dramatically increased the percentage of PBMC with markers for NK cells and produced a smaller increase in lymphocytes with surface antigens identifying them as T‐helper cells. Furthermore, retinol and retinal did not show significant change either in the percentage of lymphocytes with markers for T‐helper cells or in the helper/suppressor ratio. An increase in the expression of HLA‐Dr antigen and transferrin receptors was greater when cells were incubated with 13‐cRA than with either BC, CTX, or RBG, while carotenoids produced a greater increase in the expression of IL‐2 receptors than 13‐cRA. Our study indicates that both retinoids and carotenoids might be activating different subpopulations of immune cells.

DNA ploidy, grade, and stage in prognosis of uterine cervical cancer

A retrospective study of 56 cases of uterine cervical squamous carcinoma evaluated DNA content, histological grade, and clinical stage as indicators of prognosis. Minimum survivor follow-up was 24 months. Following standard radiation therapy, there were 40 cures and 16 treatment failures. DNA content was measured by flow cytometry of pretreatment biopsies removed from paraffin. There were 18 diploid cases and 38 aneuploid (67.9%). Aneuploid cases included 6 with very high G2-M peaks (greater than or equal to 15% of the cell sample). DNA ploidy correlation with prognosis was not statistically significant. However, both grading by a multiple parameter method (P less than 0.0133) and staging (P less than 0.0064) were significant prognostic factors. Higher grade and stage correlated with treatment failure.

The Maternal Immune Response in Coccidioidomycosis: Is Pregnancy a Risk Factor for Serious Infection?

Seven subjects with prior coccidioidal disease and three with active Coccidioides immitis infection during their first trimester were studied during pregnancy and postpartum to determine their general and antigen-specific cell-mediated immune status. All ten were white and carried their pregnancies to term without incident. Decreases in total lymphocytes and T-helper and T-suppressor subsets were noted during the third trimester, presumably secondary to an increase in plasma volume. Lymphocyte responses to the mitogens phytohemagglutinin, concanavalin A, and pokeweed were mildly decreased late in pregnancy, with significant intrasubject and intersubject variation. Responses to tetanus antigen were consistently and significantly lower as pregnancy progressed, rising above first trimester levels by 12 weeks postpartum. A similar pattern of response was noted with spherulin antigen for the seven subjects with previously demonstrated coccidioidal immunity. The three subjects with active coccidioidomycosis either failed to mount a significant spherulin immune response or demonstrated an early response that fell as pregnancy progressed. This antigen-specific immune suppression continued for up to 16 months postpartum despite the fact that there was no clinical evidence of coccidioidal activity beyond the first trimester. Thus, while all three completed pregnancy without complication, the data suggest that significantly increased maternal risk may be present when active coccidioidomycosis and pregnancy occur together. This risk may be greatest among darker-skinned individuals who become infected during the latter half of pregnancy.

The effect of in vivo dexamethasone on lymphocyte subpopulations: differential response of EAhu rosette-forming cells.

The purpose of this study was to compare the differential effect of dexamethasone on “third population” lymphocytes with its effect on B and T lymphocytes. Five human volunteers were given a single oral dose of dexamethasone (6–8 mg). Samples were evaluated prior to the drug, 5 hr after drug ingestion and 24 hr following the poststeroid sample. Surface marker studies included SIg, EAC rosettes, mouse rosettes, EAhu (Ripley) rosettes, total E-rosette-forming lymphocytes, Tμ lymphocytes, and Tγ lymphocytes. Functional studies included mitogen stimulation with phytohemagglutinin, concanavalin A, and pokeweed and antigen stimulation with herpes simplex virus and Candida albicans. At the nadir of the steroid-induced lymphopenia absolute counts for all B-cell markers were decreased. No differential effects were noted in SIg using polyvalent, IgM, IgG, κ, or λ antisera. Total T lymphocytes decreased 55% (P < 0.05). There was a differential effect on Tμ and Tγ subsets and the former were significantly decreased. Unlike other lymphocyte subpopulations, cells with high avidity Fc receptors for IgG molecules, i.e., EAhu (Ripley)-rosette-forming and Tγ lymphocytes, were unresponsive to steroid treatment. EAhu (Ripley)-rosette-forming lymphocytes increased from a baseline value of 15 to 28% after dexamethasone ingestion but absolute cell counts remained essentially unchanged with a mean baseline value of 287/μl compared with a poststeroid count of 250/μl. A rebound phenomenon was noted for B, T, Tμ, and Tγ lymphocytes (P < 0.05). Functional studies of the peripheral blood mixture of lymphocytes at the nadir of the lymphopenia showed significantly suppressed responses to both mitogens and antigens.

Lymphocyte subpopulation number and function in infancy.

Normal values for percentages of lymphocyte subpopulations and functional responses to mitogen stimulation in infancy are not well established. In the present study, lymphocyte subpopulations were examined in umbilical cord blood samples and in peripheral blood samples drawn before 7 and 24 months of age (mean age 10.4 months) from a healthy population of infants born in Tucson, Arizona. Results indicate significant increases occurred from birth to later infancy in the percentages of total T cells (CD3), T-cell subsets (CD4, CD8) and B cells (CD20). The CD4/CD8 ratio and the functional responses to ConA and PWM mitogens significantly decreased from birth to later infancy. PHA responsiveness did not show a significant change. Results from cross-sectional analyses (n=271) were supported in a smaller longitudinal subset (n=37). There were no detectable ethnic- or gender-related differences in cord blood or samples obtained in later infancy. The normal values established in this study will be useful in studies of immune-system maturation and in the clinical evaluation of newborns, infants, and toddlers suspected of either acquired or congenital immune-deficiency states.

Clinical InvestigationsThe Maternal Immune Response in Coccidioidomycosis: Is Pregnancy a Risk Factor for Serious Infection?

Seven subjects with prior coccidioidal disease and three with active Coccidioides immitis infection during their first trimester were studied during pregnancy and postpartum to determine their general and antigen-specific cell-mediated immune status. All ten were white and carried their pregnancies to term without incident. Decreases in total lymphocytes and T-helper and T-suppressor subsets were noted during the third trimester, presumably secondary to an increase in plasma volume. Lymphocyte responses to the mitogens phytohemagglutinin, concanavalin A, and pokeweed were mildly decreased late in pregnancy, with significant intrasubject and intersubject variation. Responses to tetanus antigen were consistently and significantly lower as pregnancy progressed, rising above first trimester levels by 12 weeks postpartum. A similar pattern of response was noted with spherulin antigen for the seven subjects with previously demonstrated coccidioidal immunity. The three subjects with active coccidioidomycosis either failed to mount a significant spherulin immune response or demonstrated an early response that fell as pregnancy progressed. This antigen-specific immune suppression continued for up to 16 months postpartum despite the fact that there was no clinical evidence of coccidioidal activity beyond the first trimester. Thus, while all three completed pregnancy without complication, the data suggest that significantly increased maternal risk may be present when active coccidioidomycosis and pregnancy occur together. This risk may be greatest among darker-skinned individuals who become infected during the latter half of pregnancy.

A unique malignant T-cell lymphoproliferative disorder with neutropenia simulating hairy cell leukemia

The association of neutropenia with an indolent chronic T‐suppressor cell lymphoproliferative disorder (LPD) has been well documented. The morphologic features and course suggest that this is a benign disorder. The authors studied a 58‐year‐old man with a chronic T‐cell LPD, splenomegaly, and neutropenia. Chromosomal analysis revealed multiple abnormalities which progressively increased in number as the marrow lymphocytosis became more prominent. Subsequently he developed small bowel infiltration. A splenectomy resulted in only brief improvement in the neutropenia. Immunopathologic examination of the spleen was consistent with a well‐differentiated lymphocytic lymphoma of a mature peripheral T‐cell type without subset specific markers. Granulocyte–monocyte colony (CFU‐GM) formation from the patients marrow was normal and not augmented by T‐cell depletion. Neither the patients splenic T‐cells nor serum suppressed granulopoiesis. In contrast to previous T‐LPD with neutropenia whose malignant nature has been questioned, the clinical, cytogenetic, and pathologic features and course in this case indicate a malignant lymphoid process which was effectively treated with chemotherapy. Although the histologic pattern of red pulp involvement simulated hairy cell leukemia, that diagnosis was excluded by this patients clinical, morphologic, and cytochemical features.

Cellular immune functions of adults treated with a daily, long-term, low dose of 13-cis retinoic acid.

The effects of long‐term consumption of 13‐cis retinoic acid (13‐cRA) on cellular immune functions were measured in young, adult volunteers. The retinoid was administered for 9 months at about 0.13 mg/kg/day. The mean 8AM concentrations of 13‐cRA ranged between 30 and 60 ng/ml of serum throughout the study. Corticosteroid levels in plasma decreased significantly throughout treatment, declining from 15.2 ug/dL to 9.1 mg/dL (p < 0.05). T‐cell mitogenesis stimulated by PHA or A Con A was not significantly affected, although this parameter was slightly depressed during the first 2 months of treatment. The percentage of B‐lymphocytes tended to decrease during treatment and returned to normal after cessation of 13‐cRA (p < 0.05), while the percentage of T‐cells as measured by E‐rosette and by fluorescent antibody tagging of surface antigens did not change. The percentage of non T‐cells tended to increase slightly during treatment.