Lymphokine and nonlymphokine mRNA levels in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A.

Northern and dot blotting with a panel of DNA probes were used to monitor the levels of specific mRNAs in mitogen-stimulated human T cells. The induction of IL-2 and IFN mRNAs required the synergistic action of PMA and either PHA or OKT3 mAb. In contrast, several nonlymphokine genes, the protooncogenes c-fos and c-myc, and the IL-2-R gene, were induced by either PHA or PMA alone. PHA increased the background levels of a 70 kD heat shock protein mRNA, but did not affect the observed background of c-myb mRNA. For all mRNAs that were induced, isolated CD4 and CD8 T cell subsets behaved similarly. Exogenous IL-2 had little (IFN) or no (IL-2) effect on lymphokine mRNAs, but significantly increased c-myc, IL-2-R and heat shock protein mRNAs. Therefore, the stimuli for lymphokine mRNAs differed from those required for several inducible nonlymphokine genes. IL-2 and IFN mRNAs exhibited some important similarities with c-myc, however. The levels of IL-2, IFN, and c-myc mRNA followed similar kinetics, peaking at 3 h in restimulated blasts and at 12 h in unstimulated T cells. The subsequent downregulation of lymphokine and c-myc mRNAs was retarded by cycloheximide. The induction of IL-2, IFN, and c-myc mRNAs was blocked by the immunosuppressive drug CsA, but not by the inactive analog CsH, and this block occurred at the level of nuclear transcription. Since the exogenous stimuli for lymphokine and c-myc gene expression differ, we suggest that intracellular controls must be shared to account for the similarities in their kinetics of expression and CsA sensitivity.

Cytotoxic T cells both produce and respond to interleukin 2.

Interleukin 2 (IL-2) is a T cell-derived lymphokine that serves as a cofactor for the in vitro response of T lymphocytes to antigen and plays an important role in regulating the growth and/or differentiation of these cells (1, 2). It has been postulated (2, 3) that IL-2 is produced by a discrete regulatory T cell subset, with its effects being exerted on a second, functionally distinct subpopulation of T cells. Cytotoxic T cells have been included in the IL-2-responsive subset (3). Several models of immune regulation have further assumed that the T lymphocyte pool is divided into a complex array of genetically preprogrammed T cell subtypes, each performing a specific regulatory or effector function (4, 5). However, recent results from several laboratories (6-8) have failed to support such a strict functional subdivision of the T cell pool. The availability of highly purified mouse IL-2 (1) prompted us to reevaluate the distinction, if any, between IL-2-producing and IL-2- responsive T cells. For this purpose, we resorted to a cell-cloning procedure using activated T lymphocytes that were maintained only for short periods in culture. T cell clones were tested for cytotoxic activity, responsiveness to IL-2, and for the capacity to produce IL-2 after appropriate stimulation. We found no evidence for the existence of a major functional subdivision involving these parameters among alloantigen-activated T cells: the majority of clones analyzed could perform all three functions.

Antibodies to interleukin 2. Effects on immune responses in vitro and in vivo.

Antibodies to highly purified mouse interleukin 2 (IL-2) were raised in rabbits; a 1:500 dilution of antiserum completely blocked the in vitro mitogenic effect of 10(-9) M IL-2. The antisera functioned effectively to immunoprecipitate biosynthetically labeled IL-2 and the purified immunoglobulins were useful in the construction of affinity columns for the adsorption and one-step immunopurification of IL-2. The antibodies were apparently specific for IL-2 among the lymphokines, they did not block the biological effects of IL-1, IL-3, gamma-IFN, B cell stimulating factor(s), and cytotoxic T cell differentiation factor(s). When anti-IL-2 was added to the in vitro reactions, it blocked mixed leukocyte reactions (MLR) and associated lymphocyte proliferation, the in vitro generation of cytotoxic T cells, and antibody formation as assessed by erythrocyte-specific plaque-forming cells (PFC). When injected into mice, anti-IL-2 antibodies also reduced the formation of cytotoxic lymphocytes in response to allogeneic cells, suggesting that endogenous IL-2 participates in such reactions in vivo. Taken together, the results indicate that these IL-2 antibodies will be useful adjuncts in the analysis of immune response both in vivo and in vitro.

Successful allotransplantation of mouse pancreatic islets to nonimmunosuppressed recipients.

Pancreatic islets from BALB/c (H-2d) mice are rejected within 14 days of transplantation to the kidney capsule of allogeneic, CBA/H (H-2k) recipients. Cyclophosphamide pretreatment of the islet donor reduced the intensity of the allograft response, and these islets undergo a more chronic rejection process. Islets from cyclophosphamide-pretreated donors can be cultured in a gas phase of 95% O2 and 5% CO2, provided the islets are aggregated into clusters of about 50 islets. After a culture period of 7--12 days, the islet tissue was transplanted to normal allogeneic recipients, where the tissue elicited little or no allograft response over a 3-mo observation period.

Cultured thyroid allografts induce a state of partial tolerance in adult recipient mice.

Thyroid allografts (BALB/c) prepared for transplantation by cyclophosphamide pretreatment of the donor, followed by organ culture of donor tissues for 3 weeks in a gas phase of 95% O2-5% CO2 function in normal CBA recipient mice for greater than or equal to 350 days. Up to 100 days post-transplantation, the tissue can be rejected by challenge of the recipient with 10(5) BALB/c peritoneal cells. After prolonged residence in the recipient (greater than 100, less than or equal to 350 days), only a proportion of allografts are rejected when the recipient is challenged with 10(5) followed by 10(6) peritoneal cells of donor origin. Recipients of long-term allografts are partially tolerant of BALB/c tissues. They are hyporesponsive to in vivo challenge with BALB/c spleen cells. However, lymph node cells from these animals respond essentially normally to stimulation with BALB/c spleen cells in vitro. Tolerant recipients will accept a second uncultured BALB/c allograft after a transitory rejection crisis. This crisis is not observed in the primary allograft.

PCR: how to kill unwanted DNA.

Avoidance of contamination in the PCR laboratory requires the use of strict precautions. Among these, chemical decontamination of surfaces and equipment is desirable to prevent inadvertent contamination of samples by the gloved hand and by pipettors. We have investigated the use of sodium hypochloride (Clorox), in comparison to concentrated HCl, for PCR sterilization. Ten percent Clorox was found to eliminate all ethidium bromide-stainable DNA and to prevent PCR amplification of a 600-bp DNA segment within one minute of template treatment. RNA was similarly destroyed. By contrast, even 2.0 N HCl did not destroy DNA detectable by PCR within five minutes. Because of its high efficacy, low cost and relatively low corrosiveness, we recommend the use of ten percent Clorox as a decontaminant for elimination of DNA templates in the PCR laboratory.

Vitamin and mineral supplementation in Down's syndrome.

The claim that large, nonspecific doses of vitamins and minerals improve the performance of mentally retarded children has recently reappeared in both the scientific literature and the public media. This hypothesis was examined in a double-blind, case-control study involving 20 home-reared children with Down's syndrome between 5 and 13 years of age. Children were randomly assigned by matched pairs to either a vitamin/mineral group or placebo group for an 8-month study period. No significant group differences or suggestive trends were found in any tested area of development or behavior, including intelligence (IQ), school achievement, speech and language, and neuromotor function. No group differences in appearance, growth, or health were seen. No support was found for the orthomolecular hypothesis in school-aged children with Down's syndrome.

Antiretroviral effects of deoxyhypusyl hydroxylase inhibitors: a hypusine-dependent host cell mechanism for replication of human immunodeficiency virus type 1 (HIV-1).

The HIV-1 protein Rev, critical for translation of incompletely spliced retroviral mRNAs encoding capsid elements, requires a host cell protein termed "eukaryotic initiation factor 5A" (eIF-5A). This is the only protein containing hypusine, a lysine-derived hydroxylated residue that determines its proposed bioactivity, the translation of a subset of cellular mRNAs controlling G1-to-S transit of the cell cycle. We postulated that inhibiting the hypusine-forming deoxyhypusyl hydroxylase (DOHH) should, by depleting eukaryotic initiation factor 5A, compromise Rev function and thus reduce HIV-1 multiplication. We now report that the alpha-hydroxypyridones, specifically mimosine, a natural product, and deferiprone, an experimental drug, inhibited deoxyhypusyl hydroxylase in T-lymphocytic and promonocytic cell lines and, in a concentration-dependent manner, suppressed replication of HIV-1. However, the alpha-hydroxypyridones did not affect the formation of unspliced or multiply spliced HIV-1 transcripts. Rather, these agents caused Rev-dependent incompletely spliced HIV-1 mRNA such as gag, but not cellular "housekeeping" mRNAs, to disappear from polysomes. Consequently, alpha-hydroxypyridone-mediated depletion of eIF-5A decreased biosynthesis of structural HIV-1 protein encoded by gag, measured as p24, whereas the induced formation of cellular protein like tumor necrosis factor alpha remained unaffected. By interfering with the translation of incompletely spliced retroviral mRNAs, these compounds restrict HIV-1 to the early, nongenerative phase of its reproductive cycle. In the inducibly HIV-1 expressing T-cell line ACH-2, the deoxyhypusyl hydroxylase inhibitors triggered extensive apoptosis, particularly of cells that actively produce HIV-1. Selective suppression of retroviral protein biosynthesis and preferential apoptosis of retrovirally infected cells by alpha-hydroxypyridones point to a novel mode of antiretroviral action.

Doctor's other office, an alternative to the emergency room.

Doctor's Other Office (DOO), a group of ten family physicians, offers after-hours and weekend health care to non-critical patients who might otherwise go to a hospital Emergency Room, even though their medical problems do not require expensive, elaborate facilities of the Emergency Room. Advantages to patients include less expense than the ER, care that is appropriate to their needs, and comprehensive, continuous care through dealing with physicians who will follow through with referral to a regular physician, often the patient's own family physician. DOO physician members enjoy a rational division of after-hours and weekend coverage among colleagues that permits more free time, yet assures that patients receive good quality, continuous care. The methods of operation are discussed, including personnel schedules, consultants, hospital coverage, and finances. The DOO income just meets office expenses, however, life insurance and other fringe benefits that physician members receive through the corporation aid in making this organization more financially attractive.

The Rural Health Program of Southern Monterey County.

The Rural Health Project of Southern Monterey County is attempting to demonstrate that a private group of physicians with the collaboration of the County Medical Society can responsibly and efficiently conduct a program of providing comprehensive medical care to indigent patients. Within the purposes of P.L. 89-749, the Rural Health Project is experimenting with a new way of organizing indigent health care and at the same time is providing the basis for comprehensive health planning at the local level.

Inhibition of T-cell activity by cyclosporin A.

Cyclosporin A (CyA) inhibits early events in the T-cell response. It strongly suppresses the activation of naive T cells by IL1 or IL2 and alloantigen. CyA exerts a selective effect on activated T cells. It inhibits the ability of these cells to release IL2 in response to antigen or mitogen restimulation, at concentration that have no effect on the ability of these same cell populations to respond to IL2 by proliferation. The specific effects of CyA are not limited to T cells, however, and this drug will inhibit IL1 production by lipopolysaccharide W-stimulated PU5-IR cells.

Interleukin 2 production by alloantigen (H-2) activated T cells.

H-2 alloantigen-activated T cells release Interleukin 2 in response to a secondary stimulus by either Concanavalin A or alloantigen. No detectable lymphokine is released in the absence of secondary stimulation and, in the case of alloantigen restimulation, lymphokine release is antigen-specific. Once formed, however, the lymphokine shows no antigen specificity in its action. Like the precursor of the cytotoxic T cell, the precursor of the Interleukin 2-producing T cell requires two signals for its activation; alloantigen and a source of costimulator activity. Once activated, T cells will release lymphokine in response to alloantigen alone.

Cellular requirements for production and release of the lymphocyte costimulator.

The lymphocyte costimulator (CoS) is a lymphokine required for the activation of T cell responses to H-2 alloantigens or mitogen, CoS activity is found in the supernatant medium of Concanavalin A (Con A) stimulated spleen cells, In this paper we investigate the cellular requirements for CoS production by Con A-activated mouse spleen cells. Maximal lymphokine production in response to Con A depends on a co-operative interaction between T cells and a nylon wool-adherent cell present in the spleen of nude mice. T cells appear to be the major producers of CoS activity, doing so only in response to an initial inductive stimulus supplied by nude spleen cells. The inductive stimulus is found as a soluble factor in the supernatant of Con A-activated spleen cells, and can also be provided by stimulatory (S+), but not by non-stimulatory (S-), tumour cells H-2 identical with the responding T cells. The activation of lymphokine-producing T cells is thus a two-signal process, requiring both mitogen and an additional inductive signal. Once activated, homogeneous populations of T cells will release lymphokine in response to mitogen alone.

Assistants to primary physicians in California.

The mid-level practitioner movement is no longer experimental; nurse practitioners and physician's assistants in California have proved to fill a necessary and viable professional role in the delivery of primary health care. The physician's assistant law (AB2109) and the Experimental Manpower Act (AB1503) have facilitated the training and functioning of these new health care professionals; more comprehensive laws are still needed to permit optimal utilization. National agencies for approval of teaching programs and testing of individual graduates will play an increasing role in the accreditation and certification procedures. Professional role difficulties, issues of sex and questions of delegation of responsibility are being resolved and it is hoped that a more equitable and patient-oriented system is evolving.

Evolution of a Family Nurse Practitioner Program to improve primary care distribution.

The Family Nurse Practitioner Program of the Univeristy of California, Davis, has effectively improved the distribution of primary health care manpower in medically underserved areas. This has been accomplished by selecting students, preceptors, and faculty from areas of need; decentralizing the clinical and didactic training sites; developing a competency-based, portable curriculum; and coordinating it all with a circuit-riding, institutionally based faculty.

Interleukin 2 production by both Ly2+ and Ly2- T-cell subsets.

Homogeneous T-cell populations produced by activating lymphocytes to I-, K-, or K+ D- region-encoded determinants of the mouse H-2 complex release interleukin 2 when restimulated by concanavalin A. Contrary to earlier reports on the cellular origin of the lymphokine, we find that interleukin 2 production can be either Ly2+ or Ly2- T-cell-dependent.