Cellularity

was attributed to impaired transport of glucose into immunocompetent cells, rather than to toxie effeets of alloxan.


Summary
In mice with alloxan-induced diabetes, humoral and cellular immunological reaetivity were weak. The number of leucocytes, and especially Iymphocytes, was redueed, and the weight and cellularity of Iymphatic organs were lower than in normal mice. Treatment of diabetic mice with insulin reversed morphological and funetional deficiency of the immunologieal system. Observed depression of immunological functions was attributed to impaired transport of glucose into immunocompetent cells, rather than to toxie effeets of alloxan.

Introduction
Infections are .. more frequent and more aggressive in diabetic hosts than in normal organisms. Cause for that would be impaired functional capability of the immunological system in diabetes, particularly the impairment of cellular immunity (Cattaneo. Saibene and Pozza 1976;Ptak, Czarnik and Hanczakowska 1975). Lymphocyte transformation test with phytohaemagglutinin showed reduced blastic transformation in patients who have permanently had relatively 0018-5043/78 0932-0381 $ 05.00 © 1978 Gearg Thieme Publishers high blood sugar level (MacCuish, Urbaniak, Campbell, Duncan and lrvine 1974). As to the functional capability of humoral immune response in diabetes, the data disagree. Some authors found essentially no differences in production of antibodies between normal and diabetic mice (Dolkart, Halpem and Perlman 1971), but others observed impaired production (Drachman, Rott and Wood 1966, Bates and Weiss 1941, Richardson 1933. We have followed in mice the dynamics of changes of immunity during the first four weeks after alloxan injection (Maduna and Slijeptevic, Slijep'tevic and Maduna, in press). Production of agglutinins and cellular reactivity of lymphocytes (measured by graftversus-host assay in non-diabetic hosts) were not impaired in diabetes. However, diabetes of recipients affected the graft-versus-host reactivity of lymphocytes from normal donors, in that the reactivity was significantly reduced.
Immunologicol tests. On days I, 7, 14 and 21 after alloxan injection miee were immunised by intraperitoneal injections of 0.5 ml of 10% suspension of sheep red blood cells (SRBC). Four days after immunisation, the cells that form hemolytic antibodies (plaque forming cells -PFC) were counted in the spleens of normal and diabetic mice. The method used was that described by lerne et al. (lerne, Nordin ond Henry 1963), modified for use on microscopic slides (Hrsok ond Marotti 1973). Cellular immunological reactivity of diabetic (C57BL/H) mice was assessed by grafting allogenic (RF/O) skin. Before skin grafting (on days 7, 14 and 25 after alloxan injection) diabetic mice were receiving insulin daily.
No insulin was injected after the day of skin transplantation. A group of normal mice was treated with 2 I.U. of insulin daily for 14 days, and then received the graft. No insulin was injected later on. The skin was transplanted, and the fate of the grafts assessed, as described by Boronie et al. (Boranie , OrJonie and Blo!i 1972).
Statistical analysis. The results are presented as mean va lues ± standard deviation (Siegel 1956). The significance of differences was assessed by Student's t-test, the level being set at 5% (p < 0.05).
In this work, we have followed the dynamics of Results changes in cellular elements of the blood, and changes Blood glucose. Diabetic mice have permanently had in the weight and cellularity of lymphatic organs of high glucose level in blood (350 to 660 mg%). Condiabetic mice. Functional ability of humoral and ce 1centration of glucose in alloxan mice treated with lular immunity has been assessed by counting plaque daily insulin was slightly over normal values (110 to forming cells (PFC) in the spleens, and by following 160 mg%). Normal mice were permanently hypo· rejection of allogeneic skin grafts. In order to discern glycaemic after injection of insulin (Fig. I). whether the changes were caused by direct toxic in· fluence of alloxan or by alloxan·induced deficiency of insulin, hyperglycaemia was corrected by admin· istration of exogenous insulin. Thus, the immuno· logical parameters have also been followed in alloxan· treated but normoglycaemic mice.
Blood smears. Diabetes did not influence the number of erythrocytes (Table 1). However, the number of leucocytes was significantly lower in mice which re· ceived alloxan and developed diabetes, than in nor· mal mice. Treatment with insulin restored the num· ber of leucocytes back to normal level in diabetic Materials and Methocls mice, but produced leukopenia in non-diabetic ani· mals. In alloxan·treated diabetic mice the number blood were also lower than in other groups of mice.
Diabetes. It was induoed by alloxan. The drug (gift of Prof. Organ weight and cellu/arity. Weight of the spleen K. Fe,derlin, .Ulm) \IOas dissolved im~~diatel~ befo~e u~ in and thymus was significantly lower in diabetic mice Hank s ,solutIon, ~d subsequently mJected mto tail ve~n.
than in mice from other three groups (Fig. 2). The The aßlmals recelved 100 mg of the substance per kg m a , " .
single injection. Blood glucose was measured by slightly mo. lymph nodes of dlabetlc mice were also hghter. Organ weight and cel/ulority. Seven days after alloxan in· jection mice were killed by cervical dislocation and the spleens, inguinallymph nodes and thymus were weighed on a torsion balanoe with acuity ± 0.1 mg. After that the or· gans were put into saline solution, cut into smaIl pieces, and pressed through nylon sieve; resulting oeUular suspension was aspirated three times through a thin needle (no. 20) to dispearse ceIlular clumps. The ceIls were counted in Spencer's chamber.
The cellularity of lymphatic organs is summarized in Table 2. Mice treated with alloxan had lower cell counts in a11 organs tested (spleen, thymus, Iymph nodes), than control mice. Th~ humoral immunological activity. The number of spleen cells that formed anti·SRBC antibodies as tested by the method of PFC enumeration in the spleen, was significantly lower in alloxan mice, re· gardless of the duration of the diabetic state (Table  3). Only in mice which received alloxan 1 day before Fis. 1. Glucose in blood of normal mice 0, normal mice treated with insulin -, and mice treated with alloxan, which subsequently did * or did not 0 receive insulin therapy. Table 1. Glucose in blood and the number of corpuscular elements in blood of normal mice, normal mice treated with insulin, and mice treated with aUoxan 4 days before, which subsequently did or did not receive insulin. logeneie skin lived significantly longer on diabetic mice than on non-diabetic controls (Table 4). Treatment with insulin accelerated rejection of the grafts in diabetic mice and made it become aImost as fast as that in normal, non-diabetic controls or in insulintreated non-diabetic mice.
It is worth noting that about 25% diabetic mice died  Glucose in blood (mg %) 112 ± 20 • p < 0.05 as compared with the group receiving no treatment after the skin grafting procedure (but these mice werp. not considered in results presented at the Table 4) whereas control, non-diabetic mice or diabetic mice treated with insulin all survived.

Discussion
Low number leucocytes in the peripheral blood, low weight of lymphatic organs, and low cellularity in mice treated with alloxan were apparently due to diabetic state rather than due toxic influence of alloxan. Insulin treatment, which returned blood glucose of alloxan-treated mice elose to normal levels, also resulted in reversal of changes in the lymphatic organs. Likewise, the humoral immunity of diabetic mice was suppressed as measured by PFC-response Insulin AUoxan Alloxan and (5) (5) insulin (8) 73.0 ± 4.0 13.0 ± 3.0· 63.0 ± 5.0 3.7 ± 0.5 0.5 ± 0.1· 4.7 ± 0.6 48.0 ± 15.0 4.7 ± 1.0· 24.0 ± 3.5 48 ± 11· 632 ± 141-133 ± 28 to SRBC, but in insulin-treated diabetic mice it was restored to nearly normal level. In control, non-diabetic mice, insulin even stimulated the production of PFCs. Data about humoral immunity in diabetics are controversial. Dolkart, Halpern and Perlman (1971) showed that hyperglycaemia did not suppress production of antibodies against BSA-antigens, and we found the same with S. typhimurium (Maduna and Slijepl:evic, in press), but Sood and coworkers showed impaired humoral irnmunity of mice treated with alloxan (Sood, Agarwal and Aurora 1975). Perhaps different types of diabetes were studied, or the tests for assessing the irnmunity were of various sensitivity, and tested various types of irnmunocompetent cells. In this work, Table 3. Glucose in blood and no. of PFC in the spleen of normal mice, normal mice treated with insulin, and mice treated with alloxan on various days before injection of SRBC (day 0), with and without insulin therapy. * p < 0.05 as compared with the group receiving no treatment Table 4. S w i v a l of allogeneic skin grafts on normal mice, normal mice treated with insulin, and on the mice treated with alloxan on various days before skin grafting, with and without insulin therapy. * p < 0.05 as compared with the group receiving no treatment we employed the PFC method, which reflects coop-xan injection. Deficient immune response could be ration of T and B lymphocytes and macrophages. ascribed to inadequate supply of insulin to the Wm-Cooperation was apparently impaired in mice with phatic tissue, resulting in deficient glucose metabolisn hyperglycaemia.
of immunocompetent cells. Another explanation wou take into account release of adrenal hormones due to Diabetic mice rejected allogeneic skin grafts more the stress caused by alloxan injection (Young 1969). slowly than control, non-diabetic mice. Thus, their The fist view finds support in our observation that cellular immunity was also impaired. It recovered with insulin therapy, but tendency to faster rejection treatment of diabetic state by insuh, resulting in de. crease of the concentration of glucose in blood, reof the grafts was wen in the group of stored weight and cellularity of the spleen and other mice treated with 2 I.U. of insulin daily. Ptak. Czarlymphatic organs, and returned the immunological re and Hanczakowska ( 1 9 7 5 ) that the sponse back to normal levels. This could be considen cellular immunity was defective in diabetic organism, in that the hypersensitivity to oxasolon (Ptak, Czar-as evidence that neither alloxan itself, nor the allom induced stress, suppressed the immunological respons nik and Hanczakowska 1975). and cOof treated mice. A more likely cause would be an inworkers observed suppression of cellular immunity and decrease of the number of T-lymphocytes in pa-adequate supply of glucose to lymphatic cells, in spit tients with juvenile diabetes (Catraneo, Saibene and of high glucose concentration in blood.

Pozza 1976).
A c k n o w l e d g e m e n t Our results show that impaired humoral and cellular We wish to thank Prof. Dr. M. Borani; for helpful suggestio! immune response develops relatively soon after and stimulating discussions during this research.

Introduction
Adipose tissue growth is aecompanied by either the deposition of lipid in existing fat eeUs or the fonnation of new fat eells, or both. The regulatory effeet Received: 12 July 1977Accepted: 30 Dec. 1977 of various honnones on the development of the mature adipoeyte from the pool of preeursor eells is not fuUy understood. The study of these influenees is important in understanding the development of adipose tissue in both the nonnal and the obese state.
The purpose of this study was to investigate the effeet of insulin and thyroxine on the eellularity of adipose tissue. Insulin is known to eause an inerease in adipose tissue mass (Picon 1967) and hyper-insulinism often aeeompanies exeessive adiposity (Karam, Grodsky antI Forsham 1963). However, previous results regarding the effeet of insulin on the cellularity of adipose tissue are eontradictory. Some workers have demonstrated inereased proliferation of new fat eells (Kazdova, Fabry antI Vrana 1974), while others have shown only inereased deposition of lipid in existing fat ceUs (Vost antI Hollenberg 1970;Sa/ans, Zarnowski antI Segal 1972). Excess of thyroxine on the other hand ean lead to a reduetion in adipose tissue mass