References and Notes 1. A. L. Byzov and Yu. A. Trifonov, Visiot, Res. 8, 817 (1968). 2. J. Del Castillo and B. Katz, Prog. Biophys. Biophys. Chem. 6, 121 (1956); F. Colomo and S. D. Erulkar, Fed. Proc. 29, 391 (1970). 3. J. E. Dowling and H. Ripps, Natuire (Lond.i 242, 101 (1973). 4. G. L. Walls, The Vertebrate E)ye and It. Adaptir e Radiations (Hafner. New York. 1963). 5. A. Lasansky, Philos. Tratis. R. Soc. Lotud. Ser. B Biol. Sci. 262, 365 (1971). 6. The composition of the mediumi, in milli- moles per 1000 cm:' of water, was Na+, 128; &., 2.6; Ca2+, 2; Mg'+, 2; Cl-. 112; HCO,. 20 IS. R. Fleisey, Fed. Proc. 27, 287 (1968)]. 7. D. A. Baylor and M. G. F. Fuortes. J. Physiol. (Lond.) 207. 77 (1970). 8. L. Cervetto and E. F. MacNichol. Jr.. Stcience 178, 767 (1972). 9. Two separate effects of calciumil on the nei-vous system have been described, one facilitating the release of transmitter (2) and the other depressing the excitability of net ev fibers aind nerve cells lB. Frankenhaeuser and A. L. Hodgkin, J. Physiol. (Lond.) 137, 218 (1957); J. S. Kelly, K. Krnjevic, G. Somjen, J. Neurobiol. 2, 197 (1969); G. E. Dambach and S. D. Erulkar, J. Physiol. (Lond.) 228, 799 (1973)1. In addition, calcium ions have been shown to reduce the dark current of rat pho- toreceptors [S. Yoshikamiii and W. A. Hagins. Biophys. Soc. Anna. Meet. Abstr. 11, 47a (1971)]. In Buffo marinuts an excess of cal- cium has been reported to produce a hyper- polarization of photoreceptors and to reduce the response to light [L. H. Pinto and J. E. Brown, Biol. Bull. 143, 473 (1972)]. It). D. A. Baylor. M. G. F. Fuortes, P. M. O'Bryan, J. Phi siol. (Lond.) 214, 265 (1971). HI. The experiments with high magnesium were started under the sponsorship of E. F. Mac- Nichol at the National Institute of Neuro- logical Diseases and Stroke, Bethesda. Mary- land, while L.C. was on a visiting fellowship. We thank V. Alpigiani, M. Benvenuti, G. Bottaro. B. Margheritti, and M. Morelli for their valuable technical help. S .Jtuly 1973: revised 24 September 1973 Inhibition of Cellular Mediated Immunity in Marihuana Smokers Abstract. 7The celllulatr nlediated imnunuity of 51 young chronic inarihuana s.lokers. as evaluated by the lyomphocyte response in vitro to allogeneic cells and to phytohemnagglutinin, was signiificanttly decreased anld similar to that of patients in whomn impairment of T (thy)nus derived) cell imlmnunity is known to occuir. This inhibition of blastogenesis m1ight be related to an itmpairtment of DNA synthesi,s. It has been previously reported (1) that delta-9-tetrahydrocannabinol (At- THC), a psychoactive substance of Canntabis, when administered to rodents alters their cellular mediated immune responsiveness, and it was suggested that similar changes might also occur in man. In our study the mixed lympho- cyte culture (MLC) and phytohema- glutinin (PHA) responsiveness of 51 marihuana smokers, 16 to 35 years old (median age 22), were studied. Only subjects who had used Cannlabis prod- ucts (at the exclusion of other drugs) at least once a week (average four times a week) for at least 1 year (aver- age 4 years) were selected for this nvestigation. Eighty-one healthy volunteers, 20 to 72 years of age (median age 44) were used as controls. Purified lymphocyte suspensions were prepared from fresh samples of venous blood by the Ficoll- Isopaque density gradient method (2). A microculture system was used for screening of cellular responsiveness (3). For the MLC test, 1 X 1 0- responding cells were incubated, per well, with 2 x 10;- stimulating cells pooled from a panel of ten donors, phenotypically dif- ferent [allogeneic cells in which 25 dif- ferent HL-A specificities were repre- se.nted (4) ]. For the PHA test, 2 x 10" respond- 1 FEBRUARY 1974 ing cells were incubated per well with I Ztg of purified PHA. The medium used was RPMI 1640 with penicillin, streptomycin, and glutamine, to which 25 percent aLitologous serum was added. Results are summarized in Table 2 and compared with data obtained in 60 patients with cancer, 20 patients with uremia, and 24 renal allograft recipients with iatrogenically induced immunosuppression. The mean values registered in the group of marihuana users were significantly lower than those of the normal, but much older, control group. Since an inverse cor- relation exists between cellular immu- nity, as reflected by in vitro lympho- cyte blastogenesis and aging (S), re- sults obtained in the group of mari- huana smokers may be interpreted as being indicative of cellular hypore- sponsiveness. Supporting this conclu- sion is the close similarity between the depressed MLC and PHA responsive- ness of marihuana users and that of cancer (6), uremia (7), and immuno- suppressed transplant patients in whom impairment of T (thymus derived) cell immunity is known to occur. Further- more, we observed that in vitro inhibi- tion of PHA-induced blastogenesis of normal human lymphocytes started with 1.6 1.'M THC and was complete with 20 1tM. The major psychologically active con- stituent of Cannabis sativa is A9-THC. This substance, as well as its metabo- lites, is insoluble in H.O, but is very fat soluble, and has a half-life of sev- eral days in tissues where it might exert a cumulative and pharmacological ef- fect (8). Such an effect might be re- lated in a still unknown way to the depressed cellular immune response in vitro of chronic niarihuana smokers. The effect of THC on adrenergic re- ceptors (9) might also play a role in its immunosuppressive activity, as was suggested for other drugs administered continuously over a long period (10). This inhibition of blastogenesis might result from an impairment of DNA synthesis. One of us (A.M.) sampled lymphocytes from four marihuana smokers, cultivated the cells for 72 hours, and then observed a decreased number of cells during the period of DNA synthesis (S period of the cell cycle). There was also an increased in- cidence of chromosomal breakages, Table 1. Comparative cellular mediated immunity of normal subjects, marihuana smokers, andl patients with impairment of T cell immunity. The in vitro blastogenic response of lymphocytes was studied by the MLC and the PHA tests. The incorporation rate of [3Hlthymidine of the T lymphocytes is given in counts per minute + the standard error. MLC PHA Sihbjcts No. [1lI]Thymidinc No. ["H]Thymidine tested incorporated tested incorporated (count/min) (count/min) Normal controls 81 26400 200 81 23250 210 Cancer patients Primary tumors 16 14894 + 792 16 17501 ± 124 Regional spread 23 15816 ± 420 23 13345 ± 540 Distant spread 21 8968 ± 459 21 10516 ± 580 Uremic patients 26 12001 ± 272 Transplant patients* 24 12307 ± 357 Marihuana smokersi 34 15679 ± 499 51 13779 + 169 * After I to 4 years of immuitinostippressive therapy. At least 1 year. at least once a week; no other drtug taken. 419