In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis.

Recombinant interferon beta (IFNbeta) benefits patients with relapsing remitting multiple sclerosis (MS), but the mechanisms of action are unknown. We studied in vivo immunologic effects of IFNbeta treatment and their relationship to clinical efficacy. Cytokines were measured in blood and CSF from MS patients participating in a placebo-controlled phase III clinical trial and an open-label phase IV [corrected] tolerability study of IFNbeta-1a. Additionally, immunologic studies were conducted in animals with proteolipid protein (PLP)-induced chronic relapsing experimental autoimmune encephalomyelitis. Single intramuscular (IM) injections of IFNbeta-1a (6 MIU, 30 microg) were associated with significant in vivo upregulation of interleukin-10 (IL-10) and IL-4 but not IFNgamma mRNA in peripheral blood mononuclear cells. Forty-eight hours after each IFNbeta-1a injection, serum IL-10 levels increased and remained elevated for 1 week. IFNbeta-1a recipients in the placebo-controlled phase III clinical trial showed significantly increased concentrations of CSF IL-10 after 2 years of treatment. This response correlated with a favorable therapeutic response. Exposure of PLP-reactive murine T-cell lines to IFNbeta resulted in increased antigen-driven expression of IL-4 and IL-10 and reduced encephalitogenicity. IFNbeta-1a injections induce systemic and intrathecal immunosuppressive cytokines. Myelin-specific T cells treated with IFNbeta-1a demonstrate increased immunosuppressive cytokine expression and reduced encephalitogenicity. The relationship between increased CSF IL-10 and response to therapy suggests that induction of IL-10 is a mechanism underlying IFNbeta-1a effects in MS patients.

Interferon beta induces interleukin-10 expression: relevance to multiple sclerosis.

Interferon-beta decreases the relapse rate, relapse severity, progression of neurological disability, and development of new brain lesions observed with brain magnetic resonance imaging in relapsing-remitting multiple sclerosis patients. The mechanism of action of this effect is presently unknown. This study was based on the hypothesis that immunoregulatory effects of interferon-beta may underlie its demonstrated clinical efficacy. The objective of the study was to determine the effect of interferon-beta-1a on the expression of interleukin-10, a cytokine that strongly inhibits cell-mediated immune responses. Interferon-beta-1a induced accumulation of interleukin-10 messenger RNA and protein secretion by cultured peripheral blood mononuclear cells. The observed in vitro effects were similar for healthy control subjects and multiple sclerosis patients. Intramuscular injections of interferon-beta-1a increased serum levels of interleukin-10 at 12 and 24 hours following the injection. Greater increases were induced with 12 x 10(6)-IU than 6 x 10(6)-IU injections. The effect of interferon-beta-1a was relatively specific for interleukin-10, as treatment with interferon-beta-1a did not result in accumulation of transforming growth factor-beta messenger RNA. Upregulation of interleukin-10 represents a possible mechanism of action of interferon-beta's therapeutic effect in relapsing-remitting multiple sclerosis, and has implications for therapy of other autoimmune diseases.

Calcium channel blockers inhibit cellular uptake of thymidine, uridine and leucine: the incorporation of these molecules into DNA, RNA and protein in the presence of calcium channel blockers is not a valid measure of lymphocyte activation.

An important role of transmembrane flux of calcium in lymphocyte activation has been previously demonstrated. Herein, we demonstrate that the calcium channel blockers verapamil and isradipine are able to inhibit in a concentration-dependent manner 3H-thymidine incorporation into DNA in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). However, verapamil and isradipine diminish PHA-stimulated thymidine incorporation into DNA to the same extent whether they are added at the beginning of culture or 4 h prior to completion of a 72-h culture. Thus, 3H-thymidine incorporation into DNA in the presence of verapamil or isradipine is not a valid measure of mitogen-induced lymphocyte proliferation. Similarly, verapamil and isradipine also inhibit PHA-stimulated incorporation of 3H-leucine into protein and 3H-uridine into RNA whether the drugs are added at the beginning of culture or 4 h prior to completion of 24-h cultures. There is no intracellular accumulation of 3H-thymidine, 3H-leucine, or 3H-uridine into 10% trichloroacetic acid-soluble molecules during inhibition with verapamil or isradipine, suggesting that these drugs impair the cellular uptake of these substances rather than directly inhibiting their incorporation into DNA, protein, or RNA, respectively. Since previous reports documenting the inhibitory effects of calcium channel blockers on lymphocyte proliferation have utilized 3H-thymidine incorporation into DNA to measure proliferation, we have re-examined the antiproliferative effects of these drugs by determining their effect on PHA-stimulated cell cycle progression, employing cytofluorometric analysis of propidium iodide-stained cells. When added at the initiation of culture, both verapamil and isradipine inhibited in a concentration-dependent manner PHA-stimulated cell cycle progression.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that the antiproliferative effect of verapamil on afferent and efferent immune responses is independent of calcium channel inhibition.

Calcium channel blockers are capable of inhibiting the afferent and efferent limbs of the immune responses of human peripheral blood mononuclear cells in in vitro systems. This effect is thought to be related to the ability of the calcium channel blocker to limit the transmembrane flux of calcium. We report herein that two optical enantiomers of verapamil, one (S-) which is capable of blocking the slow calcium channel and mitogen-stimulated 45Ca++ uptake into human lymphocytes, while the other (R+) is incapable of either activity, share almost identical capabilities of depressing both the afferent and efferent limbs of immunity. These observations suggest that the inhibitory effects of verapamil on various afferent and efferent immune events are, in part at least, unrelated to the inhibition of transmembrane calcium flux.

The immunosuppressive properties of enisoprost and a 5-lipoxygenase inhibitor (SC-45662).

Metabolic derivatives of arachidonic acid such as prostaglandins and leukotrienes may alter immune responses. Enisoprost (ENO), a synthetic prostaglandin E analog, inhibited the response of human peripheral blood mononuclear cells to phytohemagglutinin in a concentration-dependent fashion: .1, 1.0, and 10 micrograms/ml yielding 4.0, 21.7, and 74.3% inhibition, respectively. ENO also potently inhibited both IL-2 production (measured by ELISA) and IL-2 responsiveness (measured by CTLL-2 response to IL-2) in a concentration-dependent manner, yet did not inhibit acquisition of IL-2 receptors. ENO similarly diminished efferent immune function in a concentration-dependent fashion, as measured by inhibition of cytotoxic T cell and natural killer effector function. A 5-lipoxygenase inhibitor (5-LO), SC-45662, also inhibited mononuclear cell response to PHA in a concentration-dependent manner: 0.1, 1.0, and 10 micrograms/ml yielding 5.0, 9.7, and 79.7% inhibition, respectively. Although 5-LO potently inhibited IL-2 production, it had no effect on IL-2 responsiveness or IL-2 receptor acquisition. Like ENO, 5-LO impaired CTL and NK effector function yet was not as potent in inhibiting CTL effector function. In summary, ENO and 5-LO inhibit afferent and efferent immune function. The inhibitory effects of these drugs are not related to cytotoxicity as cell viability is maintained for 72 hr in the presence of these drugs, and the inhibitory effect is reversible when the drugs are removed. The 5-LO does not inhibit mononuclear cell responses simply by shunting the formation of arachidonic acid precursors to form inhibitory prostaglandins, since it does not impair IL-2 responsiveness in a manner similar to ENO. These two compounds may prove to have clinical utility in organ transplantation if safely achieved serum concentrations of these drugs yield in vivo immunosuppression parallel to our in vitro results.

Additive inhibition of afferent and efferent immunological responses of human peripheral blood mononuclear cells by verapamil and cyclosporine.

We have studied the effects of verapamil (0-50 microM) on the in vitro immunological function of human peripheral blood mononuclear cells in the presence or absence of cyclosporine (0-600 ng/ml). The proliferative response to phytohemagglutinin, OKT3, and alloantigens, the generation of cytotoxic T lymphocytes following allogeneic stimulation, and mitogen-induced reduction of intracellular ATP were inhibited in a concentration-dependent fashion by verapamil alone and by cyclosporine alone. When the two drugs were added to the same culture, additive inhibition was observed. A verapamil concentration of 5 microM usually reduced by at least 50% the amount of cyclosporine necessary to cause the same level of inhibition seen when no verapamil was present. The additive inhibition of the two drugs was likely not due to additive inhibition of IL-2 responsiveness, since neither drug alone inhibited the response of an IL-2-dependent T cell clone (CTLL-2) to recombinant IL-2 except at the highest concentrations tested, where a mild additive effect was noted. Nor was the additive inhibition related to an additive effect on total IL-2 receptor expression since an additive inhibitory effect on PHA-induced IL-2 receptor expression was only seen with 50 microM verapamil, while additive functional effects on mitogen- and antigen-induced proliferation and alloantigen-induced CTL generation were seen with 5 microM verapamil doses. Verapamil or cyclosporine alone inhibited IL-2 production of PHA- and phorbol ester-stimulated peripheral blood mononuclear cells--however, no additive effect was seen when the two drugs were both added to culture, probably because of the very potent inhibition by cyclosporine alone. Natural killer cell activity of human peripheral blood mononuclear cells against K562 target cells was significantly inhibited by verapamil in a concentration-dependent fashion, while cyclosporine had a more modest concentration-dependent effect. The combination of both drugs demonstrated additive inhibition. Effector function of cytotoxic T lymphocytes was modestly inhibited by either verapamil or cyclosporine alone. A combination of the highest concentrations of verapamil and cyclosporine caused an additive inhibitory effect. In summary, these data demonstrate that verapamil and cyclosporine have concentration-dependent inhibitory activities on both the afferent and efferent limbs of immunity that were additive when verapamil was used in a concentration of at least 5 microM. The additive effects are probably not related to effects on IL-2 circuitry.

Determination of reproductive maturity in the female nine-banded armadillo (Dasypus novemcinctus).

At 3-month intervals from birth to 27 months of age, 3 female armadillos were killed. The number and size of follicles greater than 202 micron were determined, plasma progesterone concentration was measured, and values were correlated with age. Blood samples were taken monthly by femoral vein puncture and plasma was analysed by radioimmunoassay for progesterone concentration. At necropsy, both ovaries were visually inspected for a corpus luteum, weighed and then processed for routine histology. The number of normal, antral follicles greater than 202 micron were counted. These follicles were arbitrarily categorized into 15 different size groups (every 77 micron). Total number of follicles greater than 202 micron varied from 15.5 +/- 1.3 at 15 months of age to 26.3 +/- 1.9 at 21 months. Follicles of a size (greater than 978 micron) most likely to ovulate were present only at greater than or equal to 9 months of age. Progesterone values remained below the adult concentration (5 ng/ml) until 15 months of age. A concentration of progesterone indicative of ovulation (approximately 10 ng/ml) occurred between 17 and 20 months of age. The findings of the present study demonstrate that the female armadillo is reproductively mature after 15 months of age.

Evaluation of simultaneous teaching of extremities in gross anatomy program.

A gross anatomy program was designed to expose medical students to all areas of the body but shortened the dissection time on the extremities by having half the class dissect either the upper or lower extremity and then study the opposite extremity already dissected by other classmates. The program has been used for six years and was evaluated via an analysis of covariance by comparing the intramural examination performance on both the dissected and undissected extremities. There was no statistical difference in the students' performances regardless of the extremity dissected. The program was also evaluated externally by student performance on Part I of the examinations of the National Board of Medical Examiners. Students (n = 191) performed at the national average (69 percent) on all gross anatomy questions and on those questions pertaining to either extremity (66 percent). The program has efficiently utilized laboratory time with no measurable change in performance by six medical classes.

Cross-sectional anatomy: preparation of teaching specimens.

Forty cross sections of a male cadaver were prepared for a Continuing Medical Education Seminar and have been used in teaching programs for the past 3 years. The sections are easy and inexpensive to construct and can be maintained with a minimal amount of effort.

Adrenalectomy has a differential effect on the ovarian response of two strains of rat.

Evidence indicates that the ovarian regulatory mechanism of different strains of rat may respond differently to adrenalectomy; this study examined that possibility. Adult female Sprague-Dawley and Holtzman rats were maintained under constant environmental. Adult female Sprague-Dawley and Holtzman rats were maintained under constant environmental conditions and each strain was divided into groups: intact; adrenalectomized; unilaterally ovariectomized; adrenalectomized and unilaterally ovariectomized for 30 days; adrenalectomized 30 days previously and then unilaterally ovariectomized followed by one oestrous cycle; adrenalectomized 30 days previously and then unilaterally ovariectomized followed by one oestrous cycle with progesterone treatment (2 mg); adrenalectomized 30 days previously and then unilaterally ovariectomized followed by one oestrous cycle with corticosterone treatment (2.5 mg/100 g). All operations and autopsies were performed at metoestrus. Chronically adrenalectomized rats shed fewer ova per ovary than intact animals. Rats that had been unilaterally ovariectomized or adrenalectomized and unilaterally ovariectomized for 30 days showed conpensatory ovulation as compared with intact rats and rats adrenalectomized for 30 days. Only the Sprague-Dawley rats that were adrenalectomized for 30 days and unilaterally ovariectomized for one oestrous cycle demonstrated compensatory ovulation. The remaining ovary in similarly treated Holtzman rats failed to compensate. Neither progesterone nor corticosterone influenced compensatory ovulation in the Sprague-Dawley rats, but both hormones caused an increase in the number of eggs ovulated in the Holtzman animals. In conclusion, the results of this study indicated that there is a strain-specific responsiveness of the ovary to adrenalectomy as assessed by compensatory ovulation.

Clomiphene-induced ovulation in the 9-banded armadillo (Dasypus novemcinctus).

8 armadillos each received 50 mg clomiphene citrate daily for 5 days. 7 of the animals showed an increase in plasma progesterone to a level indicative of ovulation (greater than 10 ng/ml) within 6 days of the last injection. In addition, the administration of clomiphene caused the urogenital smear pattern to change from dioestrous to oestrous.

Quantitative investigation of the annual pattern of follicular development in the nine-banded armadillo (Dasypus novemcinctus).

Mature, non-pregnant, female armadillos (Dasypus novemcinctus) were killed throughout the year (3-6/month, N = 54) and both ovaries were examined for follicular development. All normal and atretic follicles > 358 micrometers were counte. Total number of normal follicles remained constant from January to June but decreased by 50% in the remaining months except in October and November. Follicles > 978 micrometer (those likely to be ovulated) were found during each month but more (> 3/ovary) were present during April, May, June and October. Atretic follicles remained constant (4-6) except in July and August when the number (10-12) was doubled. A single CL was present in 2/4, 6/6, 1/6, 1/3 and 2/3 animals in July, August, October, November and December, respectively. These data confirm that armadillos ovulate in July and suggest that ovulation may also occur in some animals in late October or November.

Plasma progesterone level during delayed implantation, gestation and postpartum period in the armadillo.

Blood samples were obtained weekly from 21 pregnant armadillos during the months of June through May and analyzed by radioimmunoassay for progesterone concentration. Following ovulation and mating, the plasma concentration of progesterone remained elevated (greater than 13 ng/ml) during the 3.5 months of delayed implantation. As implantation occurred in October, the plasma concentration of progesterone increased and remained at approximately 20 ng/ml in all animals through March. Ten of the animals delivered during March and April in captivity. The date of the blood sample collection was calculated as the number of days before or after parturition. Progesterone concentration was not decreased prior to delivery and remained elevated for as long as 55 days following parturition.