Prevention of cataract development in severely galactosemic rats by the aldose reductase inhibitor, tolrestat

With a fixed time period of galactose feeding, the rate of appearance of lenticular opacities depended on the severity of galactosemia, while with a fixed amount of galactose fed, the rate was time dependent. The capacity of tolrestat, a structurally novel inhibitor of aldose reductase (AR), to control cataract development was assessed in rats fed 30-50% galactose with the diet for 7 to 277 days. In rats fed 30% galactose for 31 days, the controlling effect of tolrestat was dose dependent, and no cataracts were detected at a dose of 35 mg/kg/day. In rats given tolrestat with the diet for 14 days, then rendered severely galactosemic with a diet containing 50% galactose, and subjected to continued treatment with tolrestat at a dose of 43 mg/kg/day, no changes were detected by slit-lamp microscopy after 207 days. The preventive effect was also dose dependent. In view of the established similarity in the pathogenesis of galactosemic and diabetic cataracts, the results obtained with tolrestat support its potential for controlling cataract development in diabetics.     

Alterations of islet microvasculature in mice treated with low-dose streptozocin

Summary Islet capillary area was followed daily in mice after treatment with low-dose streptozocin (LDS), in order to elucidate the exact period during which the insular vascular bed undergoes a significant reduction. Forty C57BL6/J mice were diabetized with 5×40 mg streptozocin (STZ)/kg body wt and killed 6, 8, 9, 10, 11, 12, 15 or 18 days after the first STZ injection. Pancreases were sectioned and processed by staining for alkaline phosphatases using a method devised by Gomori. The percentage of the islet parenchymal area occupied by intra-islet capillaries was measured using a Videoplan videoanalyzer. LDS treatment did not significantly alter the islet capillary area up to day 8; the first signs of reduction were seen on days 9 and 10 (islet capillary area at days 9 and 10 respectively was 2.68% and 2.60% of controls). At day 11 a dramatic decrease in islet capillary area was seen (1.38%), which was not accompanied by a similar reduction of the islet parenchymal area. The reduction in islet capillary area continued to progress up to day 15 by which time it had achieved the lowest level (0.72%). On day 18, values remained practically unchanged.     

Alterations of islet microvasculature in mice treated with low-dose streptozocin.

Islet capillary area was followed daily in mice after treatment with low-dose streptozocin (LDS), in order to elucidate the exact period during which the insular vascular bed undergoes a significant reduction. Forty C57BL6/J mice were diabetized with 5 x 40 mg streptozocin (STZ)/kg body wt and killed 6, 8, 9, 10, 11, 12, 15 or 18 days after the first STZ injection. Pancreases were sectioned and processed by staining for alkaline phosphatases using a method devised by Gomori. The percentage of the islet parenchymal area occupied by intra-islet capillaries was measured using a Videoplan videoanalyzer. LDS treatment did not significantly alter the islet capillary area up to day 8; the first signs of reduction were seen on days 9 and 10 (islet capillary area at days 9 and 10 respectively was 2.68% and 2.60% of controls). At day 11 a dramatic decrease in islet capillary area was seen (1.38%), which was not accompanied by a similar reduction of the islet parenchymal area. The reduction in islet capillary area continued to progress up to day 15 by which time it had achieved the lowest level (0.72%). On day 18, values remained practically unchanged.     

Carboxymethylation-induced activation of bovine lens aldose reductase.

Carboxymethylation of bovine lens aldose reductase with 10 mM iodoacetate for 1 h at 25 degrees C led to a more than 4-fold increase in kcat. Carboxymethylation led to a 3- to 5-fold increase in Km NADPH and Km D-glyceraldehyde, whereas Km L-glyceraldehyde increased approx. 30-fold. Activation of the enzyme on carboxymethylation was accompanied by a decrease in the sensitivity of the enzyme to inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), sorbinil (Kii increased from 0.4 to 109 microM) and NADP (Kis increased from 0.01 to 0.03 mM), but not tolrestat. Activation of the enzyme was almost completely prevented by NADPH and to a lesser extent by DL-glyceraldehyde. Carboxymethylation of the enzyme did not result in the generation of several partially oxidized enzyme species, indicating the absence of partially carboxymethylated forms. Primary deuterium isotope effects on the reduced enzyme were consistent with a preferred ordered kinetic reaction scheme, in which hydride transfer is not rate limiting. The hydride transfer step does not seem to be significantly affected by carboxymethylation, nor do changes in the substrate binding steps seem to contribute to the observed rate enhancement. Increase in the turnover number of the enzyme on carboxymethylation appears to be due to facilitation of the isomerization of the E:NADP binary complex. The differential effect of carboxymethylation on sorbinil and tolrestat suggests distinct inhibitor sites on the enzyme, an S-site that binds sorbinil and a T-site that binds tolrestat.     

Ganglioside Treatment Modifies Abnormal Elemental Composition in Peripheral Nerve Myelinated Axons of Experimentally Diabetic Rats

Abstract: Effects of ganglioside administration on elemental composition of peripheral nerve myelinated axons and Schwann cells were determined in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats (50 days after administration of streptozocin) exhibited a loss of axoplasmic K and Cl concentrations in sciatic nerve relative to control, whereas intraaxonal levels of these elements increased in tibial nerve. These regional changes in diabetic rat constitute a reversal of the decreasing proximodistal gradients for K and Cl concentrations that characterize normal peripheral nerve. Treatment of diabetic rats with a ganglioside mixture for 30 days (initiated 20 days after the administration of streptozocin) returned proximal sciatic nerve axoplasmic K and Cl concentrations to control levels, whereas in tibial axons, concentrations of these elements increased further relative to diabetic levels. Also in the ganglioside/diabetic group, mean axoplasmic Na concentrations were reduced and Ca levels were elevated. Mixed ganglioside treatment of nondiabetic rats significantly increased axoplasmic dry weight concentrations of K and Cl in proximal sciatic and tibial axons. Schwann cells did not exhibit consistent alterations in elemental content regardless of treatment group. Changes in elemental composition evoked by ganglioside treatment of diabetic rats might reflect the ability of these substances to stimulate Na⁺,K⁺-ATPase activity and might be related to the mechanism by which gangliosides improve functional deficits in experimental diabetic neuropathy.     

An increase in superoxide dismutase counteracts islet vascular alterations in low-dose streptozocin-treated mice

A decrease in superoxide dismutase (SOD), the first cellular defence against free radicals, occurs at about the same time as the activation of macrophages within the islets of low-dose streptozocin (LDS)-treated mice. Furthermore, a decrease in the total islet capillary area also has been shown to occur by 10 days after the first streptozocin (STZ) injection and this decline in capillary area is concomitant with the activation of macrophages as is the fall in SOD. Intracellular levels of SOD have been shown to increase after administration of acetyl-homocysteine-thiolactone (citiolone); therefore, the aim of the present study was to observe any relationship between the citiolone-induced increase in SOD levels and islet microvasculature area during LDS-induced diabetes. C57BL6/J male mice were pretreated with daily intramuscular injections of 50 mg citiolone/kg body wt. for 30 days and were then rendered diabetic with 45 mg STZ/kg body wt. given for 5 days; citiolone was given until the animals were killed (days 6, 11 and 18 after the first STZ injection). Further animals were used as non-diabetic and diabetic (STZ-only) controls. The results show that LDS-treated animals when given citiolone: (1) were generally normoglycaemic; (2) had SOD levels that were higher than those of STZ-only control animals; (3) had an islet capillary area that was larger than that of LDS-treated mice. Therefore, the administration of a free radical scavenger, namely citiolone, is able partly to counteract and delay the reduction of islet vascular area and oedema formation in LDS-treated mice.     

Early macrophage infiltration in mice treated with low-dose streptozocin decreases islet superoxide dismutase levels: prevention by silica pretreatment.

It has been hypothesized that streptozocin (STZ) given in low doses for 5 consecutive days produces diabetes by induction of peroxidation phenomena similar to those induced by free radicals. Moreover, it has been demonstrated that macrophages are among the first to invade the pancreatic parenchyma and destroy islet B cells supposedly by the release of interleukin-1 that induces free radical formation. Superoxide dismutase (SOD) is a free radical scavenger present in cells, and islet B cells are known to have extremely low levels of this enzyme. Therefore, our aim was to observe SOD activity concomitantly with the appearance of intra-islet macrophages, in early diabetes induced by low-dose streptozocin (LDS). Silica-pretreated mice showed SOD values which were comparable to those found in control animals. In LDS-only-treated mice we found that SOD levels were decreased even after only 4 days from the last STZ injection and that it is at this time that the first 'recruited' macrophages appear in the islets. Moreover, the SOD levels found at this early stage (animals were still normoglycaemic and therefore not as yet diabetic) were similar to levels found by us in a previous work, in prediabetic Bio Breeding rats, thereby ascribing a crucial factor to the lowering of SOD levels even in LDS-induced diabetes.     

Capillary area in early low-dose streptozocin-treated mice.

It has been reported that vasoconstriction of intra-islet capillaries plays an important role in the initiation of the insulitis seen in the islets of Langerhans of diabetic animals. Nevertheless, only a few studies have concentrated on islet vessels. This led us to perform an experiment with the aim to compare the islet capillary area of normal untreated and multiple low-dose streptozocin (LDS) (40 mg/kg b.wt. i.p./5 days)-treated mice. In order to identify endothelial cells a method devised by Gomori, based on the fact that these cells present alkaline phosphatases on their surface, was used. Results revealed that in LDS-treated animals the capillary area per islet is significantly reduced when compared to the vascular area of controls (p less than 0.05). This could be due to a vasoconstriction phenomenon that occurs in the islet capillaries after the streptozocin administration and before the appearance of any inflammation. Our findings could demonstrate that vasoconstriction events are involved in initiation of the diabetic disease.     

Capillary area in early low-dose streptozocin-treated mice

Summary It has been reported that vasoconstriction of intra-islet capillaries plays an important role in the initiation of the insulitis seen in the islets of Langerhans of diabetic animals. Nevertheless, only a few studies have concentrated on islet vessels. This led us to perform an experiment with the aim to compare the islet capillary area of normal untreated and mulitple low-dose streptozocin (LDS) (40 mg/kg b.wt. i.p./5 days)-treated mice. In order to identify endothelial cells a method devised by Gomori, based on the fact that these cells present alkaline phosphatases on their surface, was used. Results revealed that in LDS-treated animals the capillary area per islet is significantly reduced when compared to the vascular area of controls (p<0.05). This could be due to a vasoconstriction phenomenon that occurs in the islet capillaries after the streptozocin administration and before the appearance of any inflammation. Our findings could demonstrate that vasoconstriction events are involved in initiation of the diabetic disease.     

Chorionic gonadotropin-like proteins in the obplacental giant cells of the rabbit

Diabetes mellitus was induced in 40 male C57BL6 mice by injection of a low dose of streptozocin (45 mg/kg body weight) on 5 consecutive days. Twenty four of the mice were immunosuppressed by administration of 1.5 mg FK506/kg body weight daily for 10, 15, 18 and 24 days. Administration of FK506 almost completely inhibited the streptozocin-induced islet damage, and consequently glycaemia remained normal. In FK506-treated animals any inflammatory infiltrate was very sparse and was limited to the vascular pole of the islets. Immunocytochemical results demonstrated that infiltrating cells were Ia-immunoreactive, but were not activated. Ultrastructural observations confirmed the absence of B cell necrosis and degranulation in FK506-treated mice; the few infiltrating elements encountered did not contain phagocytic vesicles or show other signs of activation.     

Kinetic studies of AKR1B10, human aldose reductase-like protein: Endogenous substrates and inhibition by steroids

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, was identified as a biomarker of lung cancer, exhibiting high sequence identity with human aldose reductase (AKR1B1). Using recombinant AKR1B10 and AKR1B1, we compared their substrate specificity for biogenic compounds and inhibition by endogenous compounds and found the following unique features of AKR1B10. AKR1B10 efficiently reduced long-chain aliphatic aldehydes including farnesal and geranylgeranial, which are generated from degradation of prenylated proteins and metabolism of farnesol and geranylgeraniol derived from the mevalonate pathway. The enzyme oxidized aliphatic and aromatic alcohols including 20α-hydroxysteroids. In addition, AKR1B10 was inhibited by steroid hormones, bile acids and their metabolites, showing IC₅₀ values of 0.03-25 μM. Kinetic analyses of the alcohol oxidation and inhibition by the steroids and tolrestat, together with the docked model of AKR1B10-inhibitor complex, suggest that the inhibitory steroids and tolrestat bind to overlapping sites within the active site of the enzyme-coenzyme complex. Thus, we propose a novel role of AKR1B10 in controlling isoprenoid homeostasis that is important in cholesterol synthesis and cell proliferation through salvaging isoprenoid alcohols, as well as its metabolic regulation by endogenous steroids.     

Prolonged fructose feeding and aldose reductase inhibition: effect on the polyol pathway in kidneys of normal rats

The effects of diets with differing carbohydrate composition on the kidney polyol pathway were investigated. The diets employed were F = fructose rich, G = glucose rich, S = cornstarch rich, and were fed for 30 days to six groups of 12 normal male Sprague-Dawley rats with and without addition of the aldose reductase inhibitor tolrestat (T). Fructose feeding resulted in higher kidney sorbitol levels (F = 0.847 +/- 0.152, G = 0.354 +/- 0.087, S = 0.207 +/- 0.041 microM/g wet wt, P less than 0.05). This was not observed in the tolrestat-treated animals (F + T = 0.182 +/- 0.024, G + T = 0.149 +/- 0.021, S + T = 0.152 +/- 0.020 microM/g wet wt). Aldose reductase activity was reduced with tolrestat administration (F = 0.0208 +/- 0.0023, F + T = 0.0048 +/- 0.0005; G = 0.0210 +/- 0.0002, G + T = 0.0059 +/- 0.0008; S = 0.0227 +/- 0.0022, S + T = 0.0062 +/- 0.0007 microU). Myoinositol levels did not differ among groups (F = 1.973 +/- 0.182, G = 2.291 +/- 0.307, S = 2.066 +/- 0.155 microM/g wet wt), but tended to increase with aldose reductase inhibition (F + T = 2.253 +/- 0.186, G + T = 2.713 +/- 0.166, S + T = 2.618 +/- 0.221 microM/g wet wt). Plasma glucose was higher in the fructose-fed rats (F = 10.78 +/- 0.55, G = 9.09 +/- 0.058, S = 9.03 +/- 0.52, F + T = 9.75 +/- 0.61, G + T = 8.42 +/- 0.64, S + T = 8.81 +/- 0.49 mM/liter). It is concluded that prolonged fructose feeding results in the accumulation of sorbitol in the kidney, caused by increased flux of glucose through the polyol pathway. This can be prevented by aldose reductase inhibition.     

Regulation of vascular smooth muscle cell growth by aldose reductase

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.     

Regulation of vascular smooth muscle cell growth by aldose reductase

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.     

Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.     

High-resolution crystal structure of aldose reductase complexed with the novel sulfonyl-pyridazinone inhibitor exhibiting an alternative active site anchoring group

The crystal structure of a novel sulfonyl-pyridazinone inhibitor in complex with aldose reductase, the first enzyme of the polyol pathway, has been determined to 1.43 angstroms and 0.95 angstroms resolution. The ternary complex of inhibitor, cofactor and enzyme has been obtained by soaking of preformed crystals. Supposedly due to low solubility in the crystallisation buffer, in both structures the inhibitor shows reduced occupancy of 74% and 46% population, respectively. The pyridazinone head group of the inhibitor occupies the catalytic site, whereas the chloro-benzofuran moiety penetrates into the opened specificity pocket. The high-resolution structure provides some evidence that the pyridazinone group binds in a negatively charged deprotonated state, whereas the neighbouring His110 residue most likely adopts a neutral uncharged status. Since the latter structure is populated by the ligand to only 46%, a second conformation of the C-terminal ligand-binding region can be detected. This conformation corresponds to the closed state of the specificity pocket when no or only small ligands are bound to aldose reductase. The two conformational states are in good agreement with frames observed along a molecular dynamics trajectory describing the transition from closed to open situation. Accordingly, both geometries, superimposed in the averaged crystal structure, correspond to snapshots of the ligand-bound and the unbound state. Isothermal titration calorimetry has been applied to determine the binding constants of the investigated pyridazinone in comparison to the hydantoin sorbinil and the carboxylate-type inhibitors IDD 594 and tolrestat. The pyridazinone exhibits a binding affinity similar to those of tolrestat and sorbinil, and shows slightly reduced affinity compared to IDD 594. These studies elucidating the binding mode and providing information about protonation states of protein side-chains involved in binding of this novel class of inhibitors establish the platform for further structure-based drug design.     

Spirohydantoin derivatives of thiopyrano[2,3-b]pyridin-4(4H)-one as potent in vitro and in vivo aldose reductase inhibitors

The 2,3-dihydrospiro[4H-thiopyrano[2,3-b]pyridin-4,4'-imidazolidine]-2',5'-dione 3 and its 7-methyl analogue 4 were synthesized and tested for their ability to inhibit aldose reductase (ALR2). To expand the structure-activity relationships, the sulfone 5 and the acetic acid derivative 7 were also prepared and tested. Compounds 3 and 4 proved to be potent ALR2 inhibitors, with IC50 values in the submicromolar range (0.96 and 0.94 microM, respectively) similar to that of sorbinil (0.65 microM). Moreover, compound 3 was found to be highly potent in preventing cataract development in severely galactosemic rats, like tolrestat, when administered as an eyedrop solution. Docking simulations of both R- and S-isomers of 3 into the ALR2 crystal structure were carried out to guide, prospectively, the design of new analogues.     

Slow Axonal Transport of Structural Polypeptides in Rat, Early Changes in Streptozocin Diabetes, and Effect of Insulin Treatment

The synthesis and transport of slowly transported polypeptides in sciatic nerves of rats was investigated by [35S]methionine pulse labeling and gel electrophoresis in control, diabetic, and insulin-treated diabetic rats. To detect very early changes diabetes was induced by streptozocin only 5 days prior to the labeling of the dorsal root ganglion cells. Fourteen days were allowed for axonal transport. In this experimental system, the neurofilament triplet is transported at an apparent velocity of 1.1 +/- 0.1 mm/day (mean +/- SD). The actin-related complex, including actin and two polypeptides of 87 kilodaltons and 37 kilodaltons, was transported at a velocity of 2.6 +/- 0.2 mm/day. For alpha- and beta-tubulin we found an apparent transport velocity of 2.2 +/- 0.1 mm/day, placing it between actin and the neurofilament triplet. The diabetic rats had a selective 32% decrease in the amount of the heaviest neurofilament subunit: 0.47 +/- 0.19% of trichloroacetic acid-insoluble radioactivity versus 0.69 +/- 0.17% in controls; 2p less than 0.05. This decrease was associated with a proximal accumulation of the two lighter neurofilament subunits. Insulin treatment of a diabetic group failed to normalize the changes of axonal transport and additional changes suggesting a hypoglycemic injury was observed.     

Pancreatic endocrine and exocrine cell ontogeny from renal capsule transplanted embryonic stem cells in streptozocin-injured mice.

In this study, we describe pancreatic cell ontogeny in renal capsule-transplanted embryonic stem cells (ES) after injury by streptozocin (STZ), showing pancreatogenesis in situ. Seven-week-old female BALB/c nude mice were treated with either a single 175- or 200-mg/kg STZ dose, a regimen that induces substantial beta-cell damage without overt hyperglycemia, and transplanted 24 hr later with 1 x 10(5) ES. Immunohistochemistry was performed on ES tissue at 15, 21, and 28 days after transplantation using antibodies against stage- and lineage-specific pancreatic markers. After 21 days, PDX-1+ pancreatic foci first appeared in the renal capsule and expressed both amylase and endocrine hormones (insulin, glucagon, and somatostatin). These foci increased in size by day 28 because of acinar and duct cell proliferation, whereas endocrine cells remained non-dividing, and made up 2-4% of ES tumor volume. PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization patterns in pancreatic foci were comparable with embryonic pancreatogenesis. A prevalence of multihormonal endocrine cells, a characteristic of adult beta-cell regeneration, indicated a possible divergence from embryonic islet cell development. The results indicate that beta-cell damage, without overt hyperglycemia, induces a process of fetal-like pancreatogenesis in renal capsule-transplanted ES, leading to beta-cell neogenesis.     

Multiple low dose streptozocin-treated rats: biochemical and morphological effects of cyclosporin A administration

Biochemical and morphological modifications occurring in the pancreas of multiple low dose streptozocin treated rats, subjected to a preventive or concomitant immunosuppressive administration of Cyclosporin A (CyA 8mg/Kg b. wt./45 days), were observed. Biochemical assays showed that CyA significantly enhances glycaemia (p less than 0.0001) and decreases serum insulin levels (p less than 0.0001), while serum creatinine was slightly increased (p less than 0.05). Morphological findings (L.M. and T.E.M.) of rats, diabetized during the CyA administration, demonstrated a variable degree of survival of B- cells (damaged and undamaged cells were noted) and areas of modified exocrine cells (extended vacuolation). Pancreases of rats, first immunosuppressed then diabetized, showed similar ultrastructural features to those of the previous group but presented a greater B-cell loss. It is evident that CyA is only moderately effective against direct cytotoxic effects of STZ, when given concomitantly, and completely fails to protect B cells when given prior to the diabetogenic drug.