Immunotherapy at clinical diagnosis of insulin-dependent diabetes An approach still worth considering

Philippe J, Missotten M: 1990. Functional characterization of a CAMP-responsive element of the rat insulin 1 gene. J Biol Chem 265:1465-1469.

Immunotherapy at Clinical Diagnosis of Insulin-Dependent Diabetes

Pictet R, Rutter WJ: 1972. Development of the embryonic endocrine pancreas. In Steiner DF, Freinkel M, eds. Handbook of Physiology, vol 1, sect 7. Washington, DC, American Physiological Society, pp 25-66.

An Approach Still Worth Considering

Ruezinsky D, Beckmann H, Kadesch T: 1991. Modulation of the IgH enhancer’s cell type specificity through a genetic switch. Genes Dev 5129-37.

Immunointervention

Paolo Pozzilli and Noel K. Maclaren diagnosis

of insulinthe

world, with the aim of inducing and maintaining clinical remission (for

Shelton KD, Franklin AJ, Khoor A, Beechem J, Magnuson M: 1992. Multiple elements in the upstream glucokinase promoter contribute to transcription in insulinoma cells. Mol Cell Biol 12:4578-4589.

example, reduction of insulin replacement therapy with normalization of metabolic

parameters}.

Several approaches

have been considered,

ranging from the use of different kinds of immunosuppression

Shieh S-Y, Tsai M-J: 1991. Cell-specific and ubiquitous factors are responsible for the enhancer activity of the rat insulin II gene. J Biol Chem 266:16,708-16,714.

to the

use of free oxygen radical scavengers.

Although

before the onset of overt hyperglycemia

should be regarded as the most

the period of latency

favorable time for any sort of immunointewention,

adjuvant treatment

at diagnosis (in addition to intensive insulin therapy) may offer clinical

Steiner DF, Chan SJ, Welsh JM, Kwok SCK: 1985. Structure and evolution of the insulin gene. Annu Rev Genet 19:463-484. Struhl K: 1991. Mechanisms for diversity gene expression. Neuron 7: 177-l 81.

at the time of clinical

dependent diabetes has been adopted in many centers throughout

advantages in the long term. Thus, maintenance function

as a result of such an approach

control and help to prevent the insurgence

in

tions.

(Trends Endocrinol

Metab

of some residual bcell

may improve

metabolic

of late diabetic complica-

1993;4: 101-l 05)

Teitelman G: 1990. Insulin cells of pancreas extend neurites but do not arise from the ectoderm. Dev Biol 142:368-379. Walker MD, Edlund T, Boulet AM, Rutter WJ: 1983. Cell-specific expression controlled by the 5’-flanking region of insulin and chymotrypsin genes. Nature 306:557-561.

1. Insulin-dependent loss of b-cell

Welsh M, Bnmstedt J, Hellerstrom C: 1986. Effects of n-glucose, L-leucine, and 2ketoisocaproate on insulin mRNA levels in mouse pancreatic islets. Diabetes 35:228231.

reduction

deficiency of the

leading

to insulin

and the metabolic

syndrome

disease.

It is now

in the

producing

commonly

number

process

(Eisenbarth

relevant

antigens

1986). Many of the have been identified,

cells. In particular, glutamic likely

to

induction

of insulin-

cells is due to an autoimmune

and most are exclusively

Whelan J, Cordle SR, Henderson E, Weil PA, Stein R: 1990. Identification of a pancreatic 8-cell insulin gene transcription factor that binds to and appears to activate cell-typespecific expression: its possible relationship to other cellular factors that bind to a common insulin gene sequence. Mol Cell

TEM Vol.4,No. 3,1993

mass

by a selective

accepted that in this type of diabetes the

Whelan J, Poon D, Weil PA, Stein R: 1989. Pancreatic &cell-type-specific expression of the rat insulin 2 gene is controlled by positive and negative transcriptional elements. Mol Cell Biol9:3253-3259.

TEM

(type 1) diabetes

(IDDM) is characterized

Weintraub HR, Davis R, Tapscott S, et al.: 199 1. The MyoD gene family: nodal point during specification of the muscle cell lineage. Science 251:761-766.

Biol 10:1564-1572.

(Baekkeskov

Points to Consider

??

present

the 65kD

isoform of

acid decarboxylase play

a

major

role

of the autoimmune

on fl

(GAD) in

is the

response

Paolo Pozzilli is at the Endocrinology Unit, Department of Medicine II, University of Rome “La Sapienza,” 00161 Rome, Italy; and Noel Maclaren is at the Department of Pathology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, 32610 FL, USA. They are Secretary General and Chairman, respectively, of the International Diabetes Immunotherapy Group.

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et al. 1990, Kaufman

1992). Whereas constituent whether

of l3 cell, it is not yet known

it is aberrantly

cross-reactive a neoantigen,

expressed

or

(Reetz et al. 199 1, DeAizpu1992). In spite of this

immune

intervention

in IDDM have a rationale Eisenbarth

and

with a foreign antigen,

rua and Harrison uncertainty,

et al.

this antigen is a normal

trials

(Wilson

and

1990), and several have been

carried out in patients with recent-onset disease (Pozzilli et al. 1991). 2. Different therapeutic approaches have been considered diagnosed

in patients

IDDM, including

with newly attempts

at

both immunosuppression

and immuno-

modulation

Skyler

(Marks

Much criticism

and

1991).

has been expressed con-

cerning the application intervention at diagnosis

of immunoof IDDM in

particular the use of cyclosporin, because of its known renal toxicity (Feutren 1988). For the first time since the introduction of insulin in 1923, however, the natural history of this disease in its first year after

101

clinical

diagnosis

has been modified

the introduction of therapy closporin (Canadian-European

by

with cyRandom-

ized Trial Group 1988). An important selection

aspect to consider

of patients

is the

for immunointer-

vention closer to the time of diagnosis (~4 weeks); this increases the rate of clinical remission

(defined as a quantitative

re-

duction in the insulin dependency with normal metabolic parameters, such as peak plasma C-peptide responses

to oral

or i.v. Sustacal) compared with patients who are treated later on (Bougneres et al. 1988). The observation that higher clini-

autoimmune process directed at pancreatic self- or autoantigens. For completeness, the genetic susceptibility for IDDM precedes the initiation of the process that causes it and invites another level of possible interventions at some future date. Such interventions given prior to clinical onset of disease have been discussed elsewhere (Muir et al. 1992). This aspect is of much concern, considering

cal remission figures (>50%) are obtainable in patients with modest symptoms and no weight loss is most interesting. This clearly suggests that if a limited amount of the p-cell mass is destroyed at the time of diagnosis when immunointervention is adopted, more frequent and longer clinical remissions can be achieved. In the light of these results, our efforts should be directed toward the early identification of diabetic patients. By doing this, effective immunotherapy may delay the clinical onset of disease, extend the “honeymoon” phase and, last but not least, reduce the insulin requirement. Still, the problem remains of what drug, combination of drugs, or other therapies (if any) should be used at the time of clinical diagnosis of IDDM, in addition to insulin, for achieving optimal metabolic control and increasing the rate of clinical remission without incurring unacceptable side effects.

that immunointervention might be implemented in an otherwise normal individual. Therefore, immunointervention should only be adopted in subjects possessing genetic and immunologic markers of the disease, with or without the loss of first-phase insulin secretion, following an i.v. glucose challenge test (Andreani et al. 1991). Nevertheless, to obtain scientifically acceptable results, only prospective studies in large family groups of susceptible individuals should be performed. Data from small studies aiming to restore abnormal immunologic parameters should be viewed with caution, as immune markers and impaired insulin secretion can fluctuate spontaneously in these subjects (Tarn et al. 1988) and may not reflect the results of an immunointervention.

??

3. The period of latency before the appearance of overt hyperglycemia should be regarded as the most auspicious time for any sort of immunointervention in humans (Muir et al. 1992). The justification for such an approach is based on data obtained from two animal models of type 1 diabetes-the BB rat (Mordes et al. 1987) and the NOD mouse (Atkinson et al. 1990, Leiter and Serreze 199 1)-which demonstrate that immunointervention before the expected onset of diabetes prevents the development of the disease. However, the exact time of onset of the autoimmune process in humans is still unknown. Furthermore, because this is a long-term therapy, it should first of all be shown that it has no harmful side effects. Earlier interventions should be more uniformly beneficial when there are marked inflamma-

102

tory changes within the islets (insulitis), circulating autoantibodies, and autoreactive T-lymphocytes, and defects of cellular immunity, but only minimal losses of pancreatic p-cell function. Still earlier lie the initiating and inductive events of the

Different Approaches of Intervention at the Time of Clinical Onset of IDDM

The goal of treatments at this phase of the disease is to prolong the period of residual b-cell function, recognized clinically as the “honeymoon” period. The toxic effects on p-cell function of glucose itself may be accentuated in cells that are targets of autoimmunity. Shah et al. (1989) have demonstrated augmented insulin reserve in patients l-2 years after diagnosis when aggressive insulin therapy was offered initially, a finding that appears to have been substantiated by Dupre et al. (personal communication). It therefore appears that intensive insulin replacement with tight control of hyperglycemia will continue to be a mainstay of treatment in the newly diagnosed IDDM patients.

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The time of clinical onset of IDDM in the natural course of B-cell destruction is also crucial for the potential success of any type of intervention. Therefore, an early diagnosis is associated with higher residual C-peptide secretion and better chances of clinical remission (Snorgaard et al. 1990, Marks and Skyler 1991). The current presentation of IDDM has changed compared with 15 years ago, and ketoacidosis is less common, suggesting that diagnosis is now often made when appreciable amounts of residual B-cell function are still preserved (Pozzilli and Andreani 1990). This factor may improve

the rate of clinical

when immunointervention (Marks and Skyler 1991).

remission is employed

f. Immunointervention trials have been initiated in the early phases of clinical disease with use of a variety of agents (Stiller et al. 1984 and 1987, Harrison et al. 1985, Feutren et al. 1986, Bach et al. 1988) in order to induce and maintain clinical remission (that is, suspension of insulin therapy with normalization of metabolic parameters) by halting the autoimmune response. Although often disappointing in the long run, results obtained with cyclosporin (CyA) (Stiller et al. 1984, Feutren et al. 1986, Bach et al. 1988) have clearly shown that this drug may increase the clinical remission rate at 12 months compared with patients receiving insulin only [reviewed in Marks and Skyler (1991)]. However, occurrence of side effects, in particular nephrotoxicity (Heering et al. 1988), and the recurrence of the disease when the drug is withdrawn, or even when continued in immunosuppressive doses, seriously limit the use of CyA in the treatment of IDDM. Two derivatives, cyclosporin G and IMM-125, may have less nephrotoxicity and could be of interest. Whereas such generalized immunotherapy is not of value for most newly diagnosed patients, it may yet have a place in IDDM treatment, since individual patients experienced prolonged “remissions.” The ability to identify prospectively the likely eventual responders at the onset would increase the utility of the approach. 2. Of some interest are the results obtained with a combination of azathioprine and prednis(ol)one therapy (Silverstein et al. 1988). Azathioprine (Imuran) is a purine antagonist that

TEM Vol. 4, No. 3, 1993

preferentially

blocks

natural

(NK) cells and cytotoxic

killer

CD8+ T-lymphocytes.

the generation

in improved

tent or by using oxygen radical gers. As a NAD precursor,

Daily doses of the

drug of 3 mg/kg (maximum, resulted

of

increases

150 mg/day)

preservation

insulin

proving

of

IDDM

pancreatectomy

scaven-

??

Directions for the 1990s

nicotinamide

synthesis,

thereby

caused

by

(Yonemura

im-

partial

et al. 1984).

The psychological diagnosis

impact

of IDDM at

is an important

sometimes

aspect

that

is not taken sufficiently

into

B-cell function over their initial year in about half of newly diagnosed IDDM

Finally, the fact that poly(ADP-ribose)polymerase inhibitors induce pan-

account. Owing to pressure from the media, it is common that patients and

patients, especially if they were older and/or still had considerable insulin secretory capacity at diagnosis. Responses

creatic p-cell regeneration suggests that this enzyme may play a role in restricting @cell replication.

their relatives are aware of progress in this area and therefore ask the physician

were

most

frequent

in those

patients

The addition

of nicotinamide

to insu-

who were rendered lymphopenic, presumably as a reflection of effective im-

lin therapy

at diagnosis,

the insulin

dose required

munosuppression

blood glucose, maintains residual p-cell function, and increases peripheral insulin sensitivity, should be viewed favora-

Children showed

induced by the agent.

below the age of 8 invariably minimal responses, perhaps

owing to the dosage cap used in the trial. Individual patients have persisted in remission for several years to date.

to normalize

bly because of its long-term the patient in preventing

benefit to diabetic

complications. Indeed, it is clinically easier to control the disease with small

3. Other approaches that have been considered for the induction of remis-

insulin doses. Moreover, this might protect from vascular damage based on the atherogenic role of insulin. Last but not

sion include agents capable of protecting @cells from cytotoxicity mediated by free

least, a low incidence of microvascular complication has been reported in patients with some residual p-cell function

oxygen radicals or nitric-oxide-mediated B-cell damage. One of the most interesting compounds available in this regard is nicotinamide, especially since preliminary studies have suggested positive effects of this agent in ICA-positive nondiabetic relatives of IDDM probands (Elliot and Chase 1991).

(Sjoberg et al. 1987). Another interesting observation in this respect is the report by Klein et al. (1989), who have

The use of a combination of immunosuppressive therapy and agents capable of inducing B-cell regeneration may be an interesting option at the time of clinical diagnosis. During the course of the cytotoxic process, the capacity of p cells to regenerate may be limited by the persistence of the autoimmune process. Therefore, therapy in the early stages of

shown protection from proliferative retinopathy in IDDM patients who still maintain p-cell function. This encourages the adoption of some sort of adjunct therapy to insulin at clinical diagnosis of IDDM. 4. Administration of an immunotoxin or an immunosuppressive agent to Tlymphocyte subsets involved in o-cell killing may be taken into consideration to stop the ongoing cytotoxicity. Immunotoxins, a conjugate of a toxin by an enzymatic method to monoclonal anti-

overt IDDM, when some b-cell mass is still present, could involve suppressing the immune response by a classic immunosuppressive drug, while at the

bodies,

are

directed

toward

antigens

expressed on the cell surface and are at present used mainly in the therapy against tumors. The use of monoclonal antibody anti-T-lymphocytes (CDS) conjugated with ricin in early phases of IDDM has

same time facilitating the replication of B cells with a compound such as nicotinamide. In the course of autoimmune B-cell damage, DNA breaks are induced and are probably accompanied by an increase in the activity of poly(ADP-

been proposed by Skyler et al. ( 199 1) in a pilot trial. A serious limitation to the use

ribose)polymerase, which uses NAD as a substitute for DNA-repairing processes, thereby reducing the NAD content of p cells required for their health and energy metabolism. This raises the possibility that IDDM may be preventable or curable at a very early stage, either by inhibiting poly(ADP-ribose)polymerase and increasing intracellular NAD con-

TEA4 Vol. 4, No. 3, 1993

which reduces

of immunotoxins is that foreign proteins induce the formation of neutralizing antibodies, thus precluding prolonged and repeated treatment. This may significantly limit the duration of therapy; however, the use of chimeric CD4 monoclonal antibodies and a panel of immunologically irrelevant toxins may resolve some of the inherent difficulties.

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whether simply

any new therapy for a cure or induction

of clinical

remission

may be applied. As this is the case, the diabetologist has the dilemma of whether to include a newly diagnosed IDDM patient in one of the several trials that at present are running in different parts of the world (Table 1) or convincing him/ her or the parents that insulin alone is still the best and only tool for achieving normoglycemia and good metabolic control. On the other hand, induction of clinical remission and reduction of the insulin dose required to keep blood glucose within the normal levels is still seen as an important result from both the physician’s and the patient’s point of view. Therefore, we think that every effort should be made to keep patients informed about novel treatments under investigation and, furthermore, physicians should give patients a chance to participate in a trial if they are interested in doing so. The cost-benefit ratio of immunointervention at diagnosis in the light of present knowledge is in our view a favorable one. Controlling the disease with low doses of insulin as a result of an immune intervention or any other p-cell protective therapy may be of long-term beneficial effect. Diabetic microangiopathy is still a major hazard for IDDM patients, and it is now accepted that good metabolic control can certainly prevent the progression toward late diabetic complications. Indeed, it is easier to control the disease with small doses of insulin, and this could be another important goal for an adjuvant intervention at diagnosis. Unfortunately, we still do not know whether patients who underwent immune intervention at diagnosis in the past will remain free from diabetic complications at a later stage of the disease, or at least show a lower incidence of such complications. Only prospective analysis of immunologically treated patients can answer this question. In conclusion, immunointervention at the time of clinical diagnosis of IDDM may offer clinical advantages in the long

103

Table 1. Ongoing immunotherapy trials reported to the International Diabetes Immunotherapy Group (IDIG) in newly diagnosed IDDM patients (courtesy of IDIG registry) Agent: Phototherapywith ultraviolet-A light of 8-methoxypsoralentreated lymphocytes Type of study: Randomized controlled trial Place: Burnett Clinical Research Unit, The Royal Melbourne Hospital, Australia Investigator: Harrison L

Agent: Short-term high-dose insulin therapy compared with conventional insulin with continued insulin therapy thereafter Type of study: Open study Place: University of London, Ontario, Canada Investigators: Dupre JI, Stiller CR, Mahon JL Agent: Low-dose cyclosporin compared with placebo with continued insulin therapy Type of study: Double-blind trial Place: University of London, Ontario, Canada Investigntors: Dupre JI, Stiller CR, Mahon JL

Agent: Nicotinamide Type ofstudy: Double-blind trials for 6 months, continued open for other 6 months Place: University of Minnesota, Minneapolis, USA Investigator: Barbosa J

Agent: CD5-plus (H65-RTA) (anti-CD5 linked to ricin-A-chain) Type of study: Double-masked, randomized, placebo-controlled trial Place: Multicenter, USA Investigators: Skyler JS, Eisenbarth GS, Palmer J, Silverstein J, Malone J, Baker L, Raskin P, Etzwiler D, Herold K, Dunn F, Hayward A, Lorenz T

Agent: Glipzide + intensified insulin therapy Type of study: Open controlled trial in children aged 1O-18 years Place: University of California, Irvine, USA Investigators: Charles MA, Selam JL

Agent: Anti-IL-2r antibody + low-dose cyclosporin A Type of study: Open study Place: Marseille, France Investigators: Vialettes B, Vague P

Agent: Low-dose thymic hormone for maintenance remission Type of study: Open controlled trial Place: University of Navarra, Pamplona, Spain Investigator: Moncada E

Agent: Immunosuppressive doses of azathioprine and/or steroids Type of study: Double-blind trial Place: University of Gainesville, Florida, USA Investigators: Silverstein J, Maclaren NK

Agent: Low-optimal-dose azathioprine for maintenance clinical remission Type of study: Open controlled trial Place: University of Navarra, Pamplona, Spain Investigator: Moncada E

Agent: Nicotinamide (IMDIAB III) Type of study: Double-blind trial Place: University of Rome “La Sapienza” and University of Rome “Cattolica,” Italy Investigators: Pozzilli P, Ghirlanda G, on behalf of the IMDIAB Study Group

Agent: Diazoxide, nicotinamide, low-dose prednisone, ascorbic acid Type of study: Controlled randomized l-year trial Place: Barbara Davis Center for Childhood Diabetes, Denver, Colorado, USA Investigators: Chase HP, O’Brien D

Agent: Thymopentin + glutathione Type of study: Open, controlled randomized trial Place: University of Palermo, Italy Investigators: Giordano C, Galluzzo A

Agent: Nicotinamide Type of study: Multicenter open controlled trial Place: University of Istanbul, Turkey Investigators: Yilmaz MT, Bukyderim S

Agent: Low-dose cyclosporin + prednisone Type of study: Open, controlled randomized trial Place: Ospedale San Raffaele, University of Milan, Italy Investigators: Secchi A, Pozza G

Agent: Thymopentin Type of study: Randomized controlled trial Place: University of Verona, Italy Investigators: Muggeo M, Betterle C

Agent: Ascorbic acid B, carotene, nicotinamide, vitamin E Type of study: Double-blind, randomized trial Place: University Hospital, Linkoping, Sweden Investigator: Ludvigsson J

Agent: Intensive insulin therapy + prednisolone at the time of spontaneous clinical remission Type of study: Open controlled trial Pluce: University of Istanbul, Turkey Investigators: Yilmaz MT, Bukyderim S

selenium,

of clinical

of

Agent: Fish oils + cyclosporin Type of study: Open pilot study Place: University Hospital, London, Ontario, Canada Investigators: Paul TL, Atkinson PA

Agent: Nicotinamide Type of study: Randomized controlled trial Place: Universita degli Studi di Bari, Italy Investigators: Ciampolillo A, Guastamacchia

Agent: Photopheresis* Type of study: Double-blind, randomized trial Place: University Hospital, Linkoping, Sweden Investigator: Ludvigsson J * Phototherapy with ultraviolet-A light of 8-methoxypsoralentreated lymphocytes

Agent: Nicotinamide vs vitamin E (IMDIAB IV) Type of study: Comparison trial Place: Universita di Roma “La Sapienza”and University of Rome “Cattolica,” Italy Investigators: Pozzilli P, Ghirlanda G, on behalf of the IMDIAB Study Group

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E, Giorgino R

TEM Vol. 4, No. 3, 1993

term. Unfortunately, at maintaining under

the age of 8 who

are seldom disease

approaches

B-cell reserve successful.

will certainly

directed in children

develop In the

IDDM

1990s the

be diagnosed

at an

earlier stage, which implies that new efforts should be made to treat not only the newly diagnosed LDDM patient but also the prediabetic subject. Prediction of IDDM in apparently normal individuals is a prerequisite for trials aimed at preventing the disease in susceptible individuals. Attempts in this direction are now well under way and, by the end of this century, should enable us to control the sequelae of the disease and, we hope, to reduce the incidence of overt diabetes. References Andreani D, Di Mario U, Pozzilli P: 1991. Prediction, prevention and early intervention in insulin-dependent diabetes. Diabetes Metab Rev 761-77. Atkinson MA, Maclaren NK, Lucchetta R: 1990. Insulitis and diabetes in NOD mice reduced by prophylactic insulin therapy. Diabetes 39:734-739. Bach JF, Feutren G, Boitard C (The CanadianEuropean Randomized Trial Group): 1988. The prospects of immunosuppression in type 1 diabetes. Adv Nephrol 17:32 l-40. Baekkeskov S, Aanstoot HJ, Christgan S, et al.: 1990. Identification of the 64K antigen in insulin-dependent diabetes as the GABAsynthesizing enzyme glutamic acid decarboxylase. Nature 347: 15 l-l 56. Bougneres PF, Care1 JC, Castano L, et al.: 1988. Factors associated with early remission of type 1 diabetes in children treated with cyclosporin. N Eng1 J Med 318:663670. DeAizpurua HJ, Harrison LC: 1992. Glutamic acid decarboxylase in insulin-dependent diabetes mellitus. Diabetes Metab Rev 8: 133147.

Harrison LC, Colman PC, Dean B, et al.: 1985. Increase in the remission rate in newly diagnosed type 1 diabetic patients treated with azathioprine. Diabetes 34: 1306-l 308. Heering P, Westhoff A, Bach D, et al.: 1988. Renin aldosterone system and renal function under cyclosporin A. Transpl Proc ZO(Supp1 3):556-562. Kaufman DL, Erlander MC, Clare-Salzer M, et al: 1992. Autoimmunity to two forms of glutamate decarboxylase in insulin-dependent diabetes. J Clin Invest: 283-292. Klein R, Klein BEK, Moss SE, et al: 1989. Epidemiologic study of diabetic retinopathy. IX. Four-year incidence and progression of diabetic retinopathy when age at diagnosis is less than 30 years. Arch Ophthalmol 107:237-243. Leiter EH, Serreze DV: 1991. Autoimmune diabetes in the non-obese diabetic mouse. Clin Immunol Immunopathol 59:323-334. Marks J, Skyler JS: 1991. Immunotherapy of type 1 diabetes mellitus. J Clin Endocrinol Metab 72~35. Mordes JP, Desemone J, Rossini AA: 1987. The BB rat. Diabetes Metab Rev 3:725-750. Muir A, Schatz MD, Maclaren NK: 1992. The pathogenesis, prediction and prevention of insulin-dependent diabetes mellitus. Endocrinol and Metab Clin of North Am 2 1: 199219. Pozzilli P, Andreani D: 1990. Type 1 diabetes at presentation: the scene changes. Diab Med 71762-763. Pozzilli P, Ghirlanda G, Gambassi G, et al.: 199 1. Overview of immunosuppressive therapy in IDDM. In Rifkin H, Colwell JA, Taylor SI eds. Diabetes 1991. New York, Elsevier. pp 1019-1022. Reetz A, Solimena H, Matteoli M, et al.: 199 1. GABA and pancreatic beta cells: colocalization of glutamic acid decarboxylase (GAD) and GABA storage and secretion. EMBO J 10:1275-1280. Shah SC, Malone JI, Simpson NE: 1989. A randomized trial of intensive insulin therapy in newly diagnosed insulin-dependent

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Feutren G: 1988. Functional consequences and risk factors of chronic cyclosporin nephrotoxicity in type 1 diabetes trials. Transplant Proc ZO(Supp14):356-66. Feutren G, Assan R, Kersenty G, et al.: 1986. Cyclosporin increases the rate of remission in insulin-dependent diabetes of recent onset. Lancet 2: 119-l 24.

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