Current Protocols in Immunology || The BB Rat as a Model of Human Insulin-Dependent Diabetes...

UNIT 15.3The BB Rat as a Model of HumanInsulin-Dependent Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) in humans is a disorder characterized bysevere hyperglycemia, weight loss, glycosuria, and ketoacidosis. These signs and symp-toms are the result of severe insulin deficiency. IDDM results from selective destructionof the insulin-producing β cells in the pancreatic islets of Langerhans in the context of aninflammatory infiltrate. Its pathogenesis is believed to be autoimmune in origin. Affectedindividuals require chronic treatment with exogenous insulin for survival. Knowledge ofIDDM advances steadily, but the disorder remains difficult to study in humans. Thediseased organ is inaccessible, and ethical considerations limit procurement of biologicalsamples and testing of treatment modalities. Use of the BioBreeding (BB) rat to modelhuman IDDM offers numerous advantages. Characteristics of diabetes in the BB ratclosely parallel those observed in human IDDM. Diabetic animals can be biopsied,autopsied, and bred to study the genetic basis of IDDM. The genetic, immunological, andenvironmental components of the disease can all be investigated under controlled condi-tions.

Two inbred lines of BB have been used in the majority of published studies using thismodel system; these rats are designated as diabetes prone (DP-BB/Wor) and diabetesresistant (DR-BB/Wor). DP-BB/Wor rats of both sexes spontaneously develop IDDMbetween 55 and 110 days of age. They are severely deficient in circulating T lymphocytesthat express the maturational alloantigen RT6. Transfusion of histocompatible normal ratT cells to young DP recipients prevents spontaneous disease provided that RT6+ T cellsbecome engrafted. In contrast, DR-BB/Wor rats have normal levels of circulating RT6+

T cells and do not develop spontaneous autoimmunity. Several intervention strategies canbe used to induce the disease in DR-BB/Wor rats. In vivo depletion of RT6+ T cells usinga cytotoxic monoclonal antibody, together with certain immunostimulatory agents, in-duces IDDM in >90% of treated animals.

The two basic protocols describe diagnosis (see Basic Protocol 1) and prevention (seeBasic Protocol 2) of spontaneous IDDM in DP-BB/Wor rats. Two alternate protocols aregiven for the induction of autoimmune disease in DR-BB/Wor rats (see Alternate Protocol1) and for the adoptive transfer of autoimmunity into histocompatible athymic WAG nu/nurecipient rats (see Alternate Protocol 2). Support protocols are also given for diagnosis ofinsulitis (see Support Protocol 1), treatment of diabetic rats with insulin (see SupportProtocol 2), and serological analysis of blood samples from sentinel rats (see SupportProtocol 3) to monitor the pathogen status of the colony.

BASICPROTOCOL 1

MAINTENANCE AND DIAGNOSIS OF SPONTANEOUS IDDM IN DIABETES-PRONE BB RATS

This protocol describes the procurement and husbandry of BB rats, diagnosis of insulin-dependent diabetes mellitus (IDDM), treatment and care of diabetic animals, and his-topathological evaluation of insulitis. The methods in this protocol are common to all themodels of IDDM in this unit. Diabetes is diagnosed clinically by documenting a sustainedincrease in blood glucose concentration. Insulitis invariably precedes the development ofdiabetes, but does not always progress to diabetes. Insulitis is diagnosed morphologicallyby assessment of mononuclear cell infiltration of pancreatic islets (see Support Protocol1). Overt hyperglycemia occurs only when >90% of the β cell mass in the pancreas hasbeen destroyed.

Copyright © 1996 by John Wiley & Sons, Inc. Supplement 19 CPI

15.3.1

Animal Modelsfor AutoimmuneandInflammatoryDisease

Materials

DP-BB rats <50 days of age (available from Dennis Guberski; see CriticalParameters and Troubleshooting)

Food (e.g., Purina), autoclavedDrinking water: acidified tap or sterile water, adjusted to pH 3.5 to 4.0 with

1 N HClClidox (Pharmacal Research Laboratories) disinfecting solution: 1 part activator, 1

part base, and 5 parts tap water, prepared freshSentinel animals: DR-BB or immunocompetent non-BB rats0.3-ml heparinized microcuvette capillary tubes (Sarstedt)Glucose reagent strips (Clinistix, Miles Labs)

Microisolator housing (see UNIT 1.2), autoclaved, and if possible in aspecific-pathogen-free (SPF) or viral-antibody-free (VAF) facility

Cages, autoclavedBedding, autoclavedWater bottles, autoclaved100- to 300-g scaleCalibrated instrumentation for measuring glucose concentration by the glucose

oxidase method (e.g., Glucose Analyzer II, Beckman, or One Touch II, Lifescan)

Additional reagents and equipment for management of immunocompromisedanimals (UNIT 1.2), serological analysis of blood samples from sentinel rats (seeSupport Protocol 3), tail vein blood collection (UNIT 1.7), diagnosis of insulitis(see Support Protocol 1), and treatment of diabetic rats with insulin (seeSupport Protocol 2)

NOTE: All materials taken into SPF or VAF animal rooms should be vacuum autoclaved,if possible. Nonautoclavable materials and the exposed surfaces of tubes, equipment, andother objects should be disinfected by spraying with Clidox disinfecting solution. Per-sonnel handling the animals should wear sterile gowns or scrub suits, bonnets, masks,gloves, and shoe covers.

Establish animal colony1. Select and obtain animals <50 days of age of a specific variant of BB rat (see Critical

Parameters)

Various colonies of both diabetes-prone (DP-) and diabetes-resistant (DR-) BB rats havebeen established worldwide. Each is identified according to the location of the productioncolony—e.g., BB rats from the NIH-sponsored resource colony at the University ofMassachusetts Medical School in Worcester are designated as BB/Wor rats and those fromEdinburgh are designated as BB/Ed.

2. House the rats in autoclaved microisolators (see UNIT 1.2), using autoclaved cages,bedding, water bottles, and food in an SPF or VAF facility. Provide the rats withacidified drinking water ad libitum. House sentinel animals in the same animal room,but not in microisolators. Use a laminar flow hood as a workstation for animalhusbandry and for experimental procedures. Frequently disinfect animal handlingareas, animal racks, and other surfaces with Clidox disinfecting solution to help keepthe facility virus free.

An SPF or VAF facility is preferred because the frequency of spontaneous IDDM in bothDP- and DR-BB/Wor rats is known to be altered by viral infection. For example, lympho-cytic choriomeningitis virus (LCMV; Dyrberg et al., 1988) can prevent diabetes in DP rats,and Kilham rat virus (KRV) can induce IDDM in DR rats (Guberski et al., 1991).

Supplement 19 Current Protocols in Immunology

15.3.2

BB Rat as Modelof Human

Insulin-Dependent Diabetes Mellitus

3. Regularly add some used bedding from random cages of BB rats to sentinel cages.Monitor the pathogen status of the animal room using blood samples from sentinelanimals housed in the same animal room (see Support Protocol 3).

Because DP-BB rats are immunocompromised by lymphopenia, it is preferable to useDR-BB rats or immunocompetent non-BB rats as sentinels.

Monitor animals for development of IDDM4. Beginning when DP-BB rats are 50 days old, weigh each rat three times weekly.

Rats are monitored for weight loss and glycosuria, either or both of which may signal thedevelopment of IDDM. Weight loss is an early indication of impending IDDM. Diagnosisis confirmed by documenting elevated blood glucose concentrations.

5. At the same time, test each rat for the presence of urinary glucose. To procure a urinesample, grasp the rat around the thorax and apply gentle pressure or stroke gentlyover the perineum, which usually produces a drop or more of fresh urine. Touch theindicator end of a glucose reagent strip to a drop of urine and look for a color changeindicative of the presence of urinary glucose.

Urine samples are more difficult to obtain from male than from female rats. Excessivepressure should be avoided because it does not aid in sample procurement and may injurethe animal.

Test blood glucose levels6. When screening of rats at risk reveals weight loss and/or glycosuria, obtain a blood

sample by venipuncture of a tail vein to test for hyperglycemia. Immediately transferthe sample to a 0.3-ml heparinized microcuvette capillary tube. Store samples on iceand measure glucose concentration as soon as possible.

The plasma glucose concentration in whole blood at room temperature declines at a rateof ∼7 mg/dl per hour.

7. Microcentrifuge capillary tubes 3 min at 4000 rpm to pellet the cellular bloodelements to the bottom of the tube, leaving the plasma fraction at the top of the tube.

The plasma fraction of diabetic animals may be lactescent, or cloudy, due to the presenceof hyperlipidemia.

8. Follow the manufacturer’s instructions for plasma glucose determinations using aBeckman Glucose Analyzer II that has been calibrated using an appropriate glucosestandard.

The Beckman Glucose Analyzer II and comparable bench-top instruments generate thehighest-precision measurements and are suitable for analyzing large numbers of samples.If a laboratory-grade glucose analyzer is not available, determine blood glucose concen-tration on whole blood using a portable blood glucose monitoring system intended forpatient care. Glucose oxidase test strips used with digital meters (e.g., One Touch II,Lifescan) provide an alternative system with little compromise in accuracy in the range of40 to 300 mg/dl. Initial cost of these hand-held meters is minimal, but reagent strips arecostly, making these systems most suitable for low-volume applications.

The diagnosis of diabetes is established when the plasma glucose concentration exceeds250 mg/dl (11.1 mmol/liter) on two consecutive days.

9. House no more than two diabetic rats per cage. Provide the animals with water adlibitum. Change the bedding frequently because diabetic animals produce more urine.

Current Protocols in Immunology Supplement 19

15.3.3


Diagnose insulitis10. If diabetic rats are to be kept >1 day after diagnosis of IDDM, start insulin therapy

(see Support Protocol 2).

All diabetic BB rats require exogenous insulin for survival.

11. Determine the extent of insulitis, the infiltration of mononuclear cells into thepancreatic islets, by examining histological sections of pancreas (see Support Proto-col 1).

ALTERNATEPROTOCOL 1

INDUCTION OF IDDM IN DIABETES-RESISTANT BB RATSDiabetes-resistant (DR)-BB rats with induced diabetes offer advantages over spontane-ously diabetic diabetes-prone (DP)-BB animals. Untreated DR rats are not lymphopenicand their T cell populations are normal with respect to phenotype and function. Treatmentsto induce diabetes do not induce severe lymphopenia. In addition, T cells from diabeticDR-BB rats adoptively transfer IDDM with no requirement for mitogen activation (seeAlternate Protocol 2). Another practical advantage is that cells can be obtained fromcohorts of young DR-BB rats treated to become diabetic at approximately the same age.This simplifies experimental design and reduces costs, because, for example, DR-BB ratstreated at 30 days old will become diabetic between 44 and 51 days of age, whereas DP-BBrats need to be housed and tested from 55 to 120 days of age to identify all potentiallydiabetic animals.

This protocol describes a method for treating 21- to 30-day-old nonlymphopenic DR-BBrats with a cytotoxic monoclonal antibody (MAb) against the T cell alloantigen RT6,together with poly(IC), to induce IDDM (Thomas et al., 1991). It is based on transientdepletion of RT6+ T cells that are known to regulate the immune system and can preventspontaneous IDDM (see Basic Protocol 2). Poly(IC) is used because IDDM occurs inDR-BB rats that have been infected with certain viruses. Poly(IC) is a synthetic double-stranded polyribonucleotide that elicits immune responses analogous to those observedduring viral infection; specifically, it induces interferon production. This treatmentinduces IDDM in >90% of both male and female viral-antibody-free (VAF) DR-BB/Worrats within 14 to 21 days.

Additional Materials (also see Basic Protocol 1)

Hybridoma DS4.23 cells (available from the Division of Diabetes, University ofMassachusetts Medical School, Worcester, Mass.)

Complete RPMI medium (APPENDIX 2) supplemented with 2% (v/v) FBS,hybridoma serum supplement, or DR-BB rat serum (RPMI-2)

Four 4- to 6-week-old DR-BB rats (available from Dennis Guberski; see CriticalParameters and Troubleshooting)

21- to 30-day old DR-BB rats of either sex (available from Dennis Guberski)Polyinosinic:polycytidylic acid, sodium salt [poly(IC), Sigma], sterilePBS (APPENDIX 2), sterile

Additional reagents and equipment for preparing MAb supernatants (UNIT 2.6),removal of lymphoid tissues (UNIT 1.9), immunofluorescence staining ofsingle-cells suspensions (UNIT 5.3), and screening and diagnosis of IDDM (BasicProtocol 1)

1. Prepare MAb supernatant from cultures of DS4.23 cells grown in complete RPMI-2medium and filter sterilize (UNIT 2.6).

DS4.23 secretes rat Ig2b anti-rat RT6.1, the RT6 allele expressed in BB rats.

2. Verify the in vivo depleting activity of each MAb preparation by treating three 4- to6-week-old DR-BB rats with 2 ml DS4.23 supernatant once daily for 3 days. Use an


15.3.4



untreated DR-BB rat as a positive control. On day 4, recover (UNIT 1.9) and analyzelymph node cells for the presence of RT6+ cells by immunofluorescence staining ofsingle cells (UNIT 5.3).

Untreated DR rats should have the normal complement of RT6.1+ T cells in lymph node(∼65%), and DS4.23-treated rats should have only 0% to 5% residual RT6+ T cells.

3. Induce IDDM in 21- to 30-day-old DR-BB rats by injecting with 2 ml DS4.23supernatant intraperitoneally daily, five times a week (Mon. to Fri.). Prepare a 1mg/ml solution of poly(IC) in sterile PBS, and inject 0.5 mg poly(IC) per 100 g ofbody weight intraperitoneally, three times a week (Mon., Wed., Fri.).

The treatment regimen should be started prior to 30 days of age because DR-BB ratsbecome resistant to IDDM induction as they mature.

If this protocol is used as part of an experiment to investigate the underlying biology ofIDDM induction rather than to generate diabetogenic cell populations, a suitable controlgroup can be generated using an isotype-matched irrelevant control antibody such as thatproduced by the hybridoma B21-2 clone. This hybridoma secretes rat IgG2b anti-mouseI-Ab,d.

4. Beginning 10 days after the initiation of treatment, monitor body weight and test forglycosuria (see Basic Protocol 1, steps 4 to 8) three times weekly. Discontinuetreatment after diagnosis of IDDM; the disease state is permanent.

Under VAF conditions, combined treatment with anti-RT6 and poly(IC) will induce IDDMin >90% of DR rats; poly(IC) alone or administered with an irrelevant control MAb inducesIDDM in ∼30% of DR rats. Mean latency to disease onset is ∼18 days after the initiationof treatment.

ALTERNATEPROTOCOL 2

ADOPTIVE TRANSFER OF IDDM

This protocol describes the adoptive transfer of insulin-dependent diabetes mellitus(IDDM) to athymic nude rats by diabetes-resistant (DR)-BB rat cells (McKeever et al.,1990). Recipients are histocompatible (RT1u) WAG nu/nu rats that are free of spontaneousautoimmune disease and lack thymus-derived T cells. Donor cells are peripheral lymphnode cells from DR-BB rats made diabetic by treatment with anti-RT6.1 antibody andpoly(IC) (see Alternate Protocol 1). The adoptive transfer of diabetes is mediated bydiabetogenic T cells from DR-BB rats, and is cell dose–dependent (Whalen et al., 1994).DR-BB rats are chosen as donors because, unlike diabetes-prone (DP)-BB donors,successful transfer of diabetes does not require large cell doses or prior in vitro activationof T cells (see Critical Parameters). The basic adoptive transfer protocol described herecan also be used to study not only those RT6− T lymphocyte subsets active in inductionof IDDM, but also RT6+ T cells involved in regulation of IDDM. Because DR-BB ratsharbor both autoreactive and regulatory T cell populations, they are suited to this purpose.

Materials

DR-BB rats treated to induce IDDM (see Alternate Protocol 1)Anti-RT6.1 MAb (e.g., supernatant from hybridoma DS4.23; see Alternate

Protocol 1)WAG nu/nu RT1u rats of either sex, 6 to 12 weeks of age (VAF breeding stock

available from the Division of Diabetes, University of Massachusetts MedicalSchool, Worcester, Mass.)

Complete RPMI medium, HEPES-buffered (APPENDIX 2)

Nylon cell strainer, sterileDisposable hand towels, sterile1-ml syringesGlass beaker, sterile


15.3.5


Gauze, sterile

Additional reagents and equipment for euthanasia by CO2 inhalation (UNIT 1.8),removing lymph nodes (UNIT 1.9), preparing single-cell suspensions (UNIT 3.1),counting cells (APPENDIX 3A), and screening and diagnosis of diabetes (see BasicProtocol 1)

NOTE: All solutions and equipment coming into contact with cells should be sterile, andproper sterile technique should be used accordingly. Injections should be performed in alaminar flow hood in the animal room, with as much adherence to sterile technique aspossible.

1. After DR-BB rats treated to induce IDDM are diagnosed, discontinue poly(IC)treatment, but continue to administer anti-RT6.1 antibody (2 ml intraperitoneallydaily, five times a week) until rats are used as donors.

Continued treatment with anti-RT6.1 antibody insures that RT6.1+ regulatory T cells, whichare continually emerging from RT6.1− thymus precursors, do not repopulate the DR donorsand thereby inhibit the adoptive transfer of diabetes.

2. Euthanize acutely diabetic RT6-depleted DR donors, using CO2 inhalation (UNIT 1.8),and harvest cervical and mesenteric lymph nodes (UNIT 1.9).

3. Prepare a single-cell suspension from donor lymph nodes (UNIT 3.1). Adjust the cellconcentration to 1.0 × 107 cells/ml in sterile HEPES-buffered complete RPMImedium. Filter through a sterile nylon cell strainer, and keep cells cold until they areinjected into recipients.

Pooled lymph nodes from a single treated diabetic DR donor should yield ∼6–8 × 107 cells,enough cells to treat six to eight recipient rats. Adoptive transfer of IDDM is T cell mediatedbut does not require purification of T cells prior to transfer.

4. Wrap a 6- to 12-week old WAG nu/nu RT1u recipient rat securely in a steriledisposable towel, covering the head and leaving the tail exposed.

This calms the animal and assures restraint during the injection. It is not necessary to holdthe rat tightly if it is properly wrapped.

5. Hold the rat around its chest and immerse the tail for 10 sec in a sterile glass beakerof warm water to dilate the veins. Apply gentle pressure at the base of the tail to causethe veins to tumesce.

The most rapid and successful injection procedure requires two individuals to perform, butavoids the stress of anesthesia. One person secures the recipient rat and a second personperforms the injection.

6. Using a sterile 1-ml syringe, inject 1 ml of the cell suspension (1 × 107 viable cells)slowly into each recipient then retract the needle. Compress the injection site with asterile gauze pad until bleeding stops.

Either a 1-ml insulin syringe with a 28-G needle or a 1-ml syringe with the needlereplaced by a butterfly infusion catheter with a 25-G needle can be used to inject thecells. With an insulin syringe, the entire sample will be ejected when the plunger isdepressed. With an infusion catheter, a volume of cells will remain in the cathetertubing, and the tubing must be flushed with sterile medium to ensure delivery of theentire volume of cells.

7. Beginning 14 days after cell transfusion, screen all recipients for IDDM by checkingfor weight loss and glycosuria (see Basic Protocol 1, steps 4 to 8).

A dose of 1 × 107 lymph node cells from an acutely diabetic DR-BB rat will induce diabetesin >90% of recipients, with a latency to onset of 18 to 24 days.


15.3.6



Adoptive transfer using this protocol can also be achieved using spleen cells from acutelydiabetic DR-BB rats in place of lymph node cells (McKeever et al., 1990). Thymocytes fromeither diabetic or untreated DR-BB rats will also adoptively transfer IDDM (Whalen et al.,1995). Two modifications of the protocol are required. A dose of ≥2 × 108 thymocytes shouldbe used to achieve >85% transfer. In addition, all recipients must be treated with anti-RT6.1MAb to preclude the expansion of RT6+ regulatory T cells from the thymocyte inoculum. Ifthe experiment is performed under VAF conditions, recipients must also be treated withpoly(IC) (see Alternate Protocol 1).

BASICPROTOCOL 2

PREVENTION OF IDDM IN DIABETES-PRONE BB RATS

Spontaneous diabetes in diabetes-prone (DP)-BB rats can be prevented by a singletransfusion of T cells from the peripheral lymphoid tissues of histocompatible nonlym-phopenic rat strains. Prevention by this method requires early intervention, before theonset of spontaneous insulitis; it is generally ineffective after 60 days of age (Burstein etal., 1989). Engraftment of RT6+ T cells, which are absent in the unmanipulated DP-BBrat, is required for protection from disease. In this protocol, 21- to 30-day-old DP-BB ratsare administered unfractionated spleen cells from coisogenic diabetes-resistant (DR)-BBrats. Although T cells mediate protection from IDDM, purification of T cells prior totransfer is not required. Because RT6 is a maturational alloantigen that is not fullyexpressed in young rats, donor DR rats are used at 8 weeks of age or older, when thecohort of RT6+ T cells is fully developed. The degree of protection is a function of thenumber of cells transfused; >90% protection is achieved with a single dose of 2 × 108 DRspleen cells. This procedure is often used to facilitate breeding in DP-BB rat colonies.Mating diabetic male DP-BB rats with female DP-BB rats that have been prophylacticallytransfused to prevent diabetes enhances both fertility and the adequacy of subsequentnursing (Guberski et al., 1992).

Materials

21- to 30-day old DP-BB rats of either sex (available from Dennis Guberski; seeCritical Parameters and Troubleshooting)

8- to 12-week old DR-BB rats of either sex (available from Dennis Guberski)Complete RPMI medium, HEPES-buffered (APPENDIX 2)

Nylon cell strainer, sterile1-ml syringes with 23-G needles

Additional reagents and equiment for euthanasia (UNIT 1.8), removing the spleen(UNIT 1.9), preparing a single-cell suspension (UNIT 3.1), counting cells(APPENDIX 3A), intraperitoneal injections (UNIT 1.6), and screening and diagnosisof diabetes (see Basic Protocol 1)

NOTE: All solutions and equipment coming into contact with cells should be sterile, andproper sterile technique should be used accordingly. Injections should be performed in alaminar flow hood in the animal room, with as much adherence to sterile technique aspossible.

1. Randomly divide 21- to 30-day-old DP-BB rats into two groups: one for transfusionwith DR-BB rat spleen cells, and the other to be used as an untreated control groupof spontaneously diabetic rats.

Transfusion recipients should be treated when ≤30 days of age.

2. Euthanize one 8- to 12-week-old DR-BB rat for each recipient DP-BB rat (UNIT 1.8).Remove the spleen (UNIT 1.9) and prepare a single-cell suspension of spleen cells(UNIT 3.1).


15.3.7


3. Resuspend cells to a final concentration of 2 × 108 cells/ml in HEPES-bufferedcomplete RPMI medium. Filter through a sterile nylon cell strainer and keep cellscold until used.

It is not necessary to remove erythrocytes.

4. Inject each recipient in the transfusion group with 1 ml spleen cell suspension, eitherintraperitoneally or intravenously.

Both routes of administration are effective.

5. When the DP-BB rats reach 50 days of age, begin screening the animals in both thetransfusion and the control groups for weight loss and glycosuria (see Basic Protocol1, steps 4 to 8), and continue screening until IDDM develops or until the animalsreach 120 days of age.

After 120 days of age, the probability of spontaneous diabetes is <20% in a VAF colony.The group of untreated control DP-BB rats will document the frequency of spontaneousdisease.

Previous studies have shown that DR-BB/Wor and Wistar Furth spleen cells are equallyefficacious in preventing IDDM in DP-BB rats and can be given either intraperitoneallyor intravenously. Lymph node cells, which have a higher frequency of RT6+ T cells, canalso be used to protect DP-BB rats. Donor cell engraftment can be documented, if desired,by immunofluorescence staining for RT6+ cells, which are nearly absent in the peripherallymphoid tissues of untreated DP-BB rats.

SUPPORTPROTOCOL 1

DIAGNOSIS OF INSULITIS

Insulitis is diagnosed morphologically by documenting the appearance of mononuclearcell infiltrates into pancreatic islets. Infiltrates may be observed in the islets (insulitis)and also adjacent to islets (periinsulitis), in perivascular areas, and in the exocrinepancreas. Islets from diabetic rats always exhibit extensive lymphocytic insulitis and/orend-stage islet characteristics (see step 7). To diagnose insulitis, whole pancreases areremoved, fixed, processed for histological assessment, and evaluated by light microscopy.

Materials

DR-BB or DP-BB ratBouins fixative (see recipe)10% (v/v) buffered formalin (Baxter)

Dissecting instrumentsTissue-processing histology cassettes (Fisher)Histology pencil (Fisher)

Additional reagents and equipment for CO2 euthanasia (UNIT 1.8)

1. Euthanize the rat in an atmosphere of 100% CO2 (UNIT 1.8).

2. Open the abdomen. Grasp the spleen with forceps and withdraw the spleen andattached pancreas from the peritoneal cavity. Use scissors to free the pancreas fromthe duodenum. Remove the pancreas and attached spleen to a flat surface and use finescissors to separate the pancreas from the spleen.

The pancreas of the rat is a diffuse whitish organ with multiple lobes. The pancreas isattached at one end, the head, to the inner radius of the duodenum and at its other end, thetail, to the spleen. Removing the pancreas in this manner prevents confusing the organ withfatty omental tissue.


15.3.8



3. Place the pancreas into a histology cassette and gently spread it with fine forceps tomaximize surface exposure to the fixative. Label the cassette on the writing surfacewith a histology pencil using a coding system to permit blind processing and scoringof samples.

4. Close the cassette and place it immediately in a plastic or glass container containingBouins fixative. Immerse cassettes completely. Leave in Bouins fixative overnight orup to 1 day.

Fixation in Bouins for >24 hr will cause the tissue to become brittle and difficult to section.

5. Rinse 8 to 24 hr with a continuous gentle stream of tap water. After rinsing, store thecassette completely submerged in 10% buffered formalin until processed.

Samples can be stored in formalin for extended periods if necessary.

6. Submit fixed tissues to a histology or pathology laboratory; alternatively, paraffinembed the tissue and mount two to three 5-µm sections from each pancreas onmicroscope slides. Stain the sections with hematoxylin/eosin for analysis.

It is recommended that fixed tissues be submitted to a histology or pathology laboratoryfor processing.

Hematoxylin/eosin stains nuclei blue to purple and cytoplasmic structures in lighter pinkshades. Islets of Langerhans are scattered throughout the exocrine pancreas and appearas circumscribed islands of tissue that stain a lighter pink than the acinar tissue. The nucleiof infiltrating mononuclear cells appear blue-purple against the lighter pink backgroundof the endocrine and exocrine pancreas.

7. Evaluate pancreas sections for insulitis using a light microscope.

Slides should be evaluated by individuals who are familiar with normal and abnormalpancreatic morphology and who are unaware of the treatment status of the tissue donor.For quantitative analyses, each pancreas should be scored independently by two individu-als for the presence and intensity of insulitis.

A normal pancreas has abundant islets and no cellular infiltrates. Early insulitisappears before the onset of hyperglycemia. As inflammation progresses, both thenumber of affected islets and the density of the infiltrate within the islets increase. Asthe central core of β cells within an islet is destroyed, the lymphoid cell infiltratedisappears and the residual islet α, δ, and pancreatic polypeptide-secreting cells forma shrunken, misshapen structure termed an end-stage islet (Nakhooda et al., 1977).Immunohistochemical and other methods have shown the cells comprising the insulitislesion include CD4+ and CD8+ T lymphocytes, natural killer (NK) cells, macrophages,and, to a lesser extent, B lymphocytes. The earliest infiltrating cell type may bemacrophages.

SUPPORTPROTOCOL 2

INSULIN THERAPY FOR DIABETIC RATS

Two methods of insulin therapy are described: daily subcutaneous injections of insulinfor short-term maintenance and subcutaneous implantation of timed-release pellets ofbovine insulin for long-term maintenance. The goal of treatment is not to produce a normalblood glucose concentration, as such “tight” control poses the risk of death fromhypoglycemia, but rather to maintain growth and prevent ketoacidosis for the duration ofthe experiment. The dose of insulin is chosen on this basis. It is not necessary to monitorblood glucose concentrations in treated animals unless this parameter is a variable in anexperimental study.

Materials

Diabetic rats (see Basic Protocol 1 or Alternate Protocol 1 or 2)Glucose reagent strips (Clinistix, Miles Labs)


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U-100 Ultralente insulin, bovine or porcine (Novo Nordisk Bioindustrials)Insulin diluent (Novo Nordisk Bioindustrials)Linplant sustained-release bovine insulin implants and standard 12-G trocar/stylet

(Linshin Canada)Ketone reagent strips (Ketostix, Miles Labs)Lactated Ringers solution (Baxter), sterile0.5-ml U-100 insulin syringes, sterile

Additional reagents and equipment for anesthesia by methoxyflurane inhalation(UNIT 1.4) and subcutaneous injection (UNIT 1.6)

1. Weigh diabetic rat and monitor for glycosuria daily using glucose reagent strips.

Weight is used to determine the insulin dose and to monitor the adequacy of therapy. Anadequate dose will prevent weight loss, dehydration, and diabetic ketoacidosis.

For short-term insulin therapy2a. Dilute Ultralente U-100 insulin with an equal volume of sterile insulin diluent to give

a final concentration of 50 U/ml.

Insulin should be protected from extremes of temperature; in a laboratory setting it isprudent to refrigerate it.

All currently available insulins are supplied at a concentration of 100 U/ml (“U-100").Because the appropriate dose for a rat is very small (∼1 U/100 g body weight), the insulinshould be diluted to 50 U/ml to facilitate accurate dosing.

3a. Resuspend the insulin by rolling the bottle between the hands. Using an 0.5-ml U-100insulin syringe, inject a volume of air equal to the insulin dose into the bottle of dilutedUltralente insulin and withdraw the required volume of insulin, making sure thereare no air bubbles. Inject diabetic rat subcutaneously once daily.

A dose of 1 U/100 g body weight is suitable for most animals. Because rats are nocturnalfeeders, insulin therapy is best administered late in the day to ensure adequate insulin levelsduring the evening period of activity and feeding.

Injection may be into any subcutaneous site; the nape of the neck and the chest are easiest.To inject into chest skin, grasp the rat around the thorax from behind, pinching a fold ofskin between thumb and forefinger. With the rat facing you, inject the insulin subcutane-ously.

Treatment with a single daily injection of insulin is recommended when maintenance foronly short periods is required. Ultralente insulin of animal rather than human origin (e.g.,recombinant human insulin, Eli Lilly) is recommended for treatment of all diabetic rats.The authors have observed empirically than animal insulins provide better control ofdiabetes in the rat than do human insulins.

For long-term insulin therapy2b. Anesthetize diabetic rat with methoxyflurane (UNIT 1.4).

3b. Following manufacturer’s directions for insertion of implants, insert a Linplanttimed-release insulin pellet subcutaneously into the flank or nape of the neck of therat.

The number of pellets is determined by the rat’s weight. Use one-half of a pellet for rats100-150 g, and one entire pellet for larger rats. A wound clip is not necessary to close thesmall incision site.

These pellets reliably provide ∼2 U of insulin per day for 45 to 60 days. They are veryuseful for long-term maintenance of diabetic animals. They can stabilize blood glucose


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concentration more effectively than single daily doses of insulin, but they are generally notused for short-term treatment because they are costly.

4. Daily, weigh and monitor for glycosuria each rat maintained on chronic insulintherapy. If weight declines and a high level of urinary glucose is present, measureurinary ketone levels using the ketone reagent strips.

Ketoacidosis is the result of loss of control of diabetes and can be fatal. The presence ofketones is indicated by a color change in the test strip.

Weight loss, with or without ketoacidosis, usually indicates that the daily insulin dose needsto be increased—an additional 0.5 U per day generally suffices. Too much insulin can resultin hypoglycemia and death. Occasionally, weight loss, hyperglycemia, and ketoacidosisresult from an error in insulin injection, failure of a pellet, or some form of stress.

5. If urinary ketones are present, administer 5 to 10 ml sterile lactated Ringers solutionsubcutaneously into the nape of the neck to compensate quickly for the urinary fluidlosses that accompany ketoacidosis.

SUPPORTPROTOCOL 3

SEROLOGICAL ANALYSIS OF BLOOD SAMPLES FROM SENTINEL RATS

The pathogen status of the rats is most easily monitored by serological analysis of serumsamples from sentinel rats, screening for the presence of serum antibodies to a panel ofviral and bacterial microorganisms. Additionally, the animals should be routinelyscreened for common parasites (UNIT 1.1).

Materials

PBS (APPENDIX 2)

Additional reagents and equipment for anesthesia by methoxyflurane inhalation(UNIT 1.4), tail vein or orbital plexus blood collection (UNIT 1.7), and preparationof serum from blood (UNIT 2.4)

1. Anesthetize each sentinel rat using methoxyflurane inhalation (UNIT 1.4).

2. Collect ∼1 ml blood from the tail vein or orbital plexus (UNIT 1.7) of anesthetizedsentinel rat into a 1.5-ml microcentrifuge tube.

3. Allow blood to clot (UNIT 2.4).

4. Microcentrifuge tubes 10 min at 4000 rpm to pellet cells and collect serum foranalysis.

5. Dilute 200 µl serum 1:5 in PBS and submit to Charles River Laboratories (seeAPPENDIX 5) or one of the government-sponsored laboratories listed in Table 1.1.2 forcomplete serological assessment for rats.

A complete assessment includes screening for Sendai virus, pneumonia virus of mice(PVM), sialodacryoadenitis virus (SDAV), rat coronavirus, Kilham rat virus (KRV), H-1virus, GD7, Reovirus-3, Mycoplasma pulmonis, lymphocytic choriomeningitis virus(LCMV; see UNIT 19.9), mouse adenovirus, Hantaan virus, Encephalitozoon cuniculi, andcarbacillus. Monthly serological assessment of sentinel animals is recommended.

A new rat parvovirus with antigenic similarity to KRV) has recently been identified anddesignated RPV-1 (Jacoby and Ball-Goodrich, 1995). The RPV-1 status of the colonyshould also be monitored. At this writing, this can be done by requesting an additionalimmunofluorescence analysis for KRV; the presence of RPV-1 is indicated by a negativeELISA test for KRV and a positive immunofluorescence test for KRV.

Current Protocols in Immunulogy Supplement 37

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REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2; for suppliers, see APPENDIX 5.

Bouins fixativePrepare a stock of 1 part saturated solution of picric acid and 3 parts 10% (v/v)buffered formalin (Baxter). Store in a brown glass bottle at room temperature. Justprior to use, make Bouins fixative by mixing 95% (v/v) picric acid/formalin solutionwith 5% (v/v) glacial acetic acid.

CAUTION: Wear gloves and eye protection when handling these solutions and prepare themin a fume hood.

COMMENTARY

Background InformationThe BB rat strain was derived from a colony

of outbred Wistar rats that developed spontane-ous diabetes mellitus at the BioBreeding Labo-ratories, Ottawa, Canada, in the 1970s. Affectedanimals became the founders of the inbreddiabetes-prone (DP)-BB/Wor rat strain used inthe majority of published studies. At the sixthgeneration of inbreeding, a subpopulation ofnondiabetic DP-BB/Wor rats was selected tostart a control line. Now designated as diabe-tes-resistant (DR)-BB/Wor rats, these coiso-genic descendants of DP-BB/Wor forbearers donot develop spontaneous diabetes. BothBB/Wor rat lines are now fully inbred. BB/Worand all other BB rat strains express the RT1u ratmajor histocompatibility complex (MHC)haplotype. As is the case with human insulin-dependent diabetes mellitus (IDDM), predis-position to the disease is strongly linked geneti-cally to MHC genes. The onset of hypergly-cemia is preceded by mononuclear cellinfiltration of the islets (insulitis) and the ap-pearance of various autoantibodies directedagainst islet cells, insulin, and other tissues.Onset of hyperglycemia is associated withweight loss, glycosuria, and ketosis. Exoge-nous insulin therapy is required from the timeof disease onset, or death ensues rapidly fromketoacidosis.

Although both lines of BB/Wor rats are usedto model human IDDM, there are importantdifferences between DP and DR-BB/Wor rats.DP-BB/Wor rats have severe T cell lym-phopenia associated with the lyp gene. Thelymphopenia results in a deficiency in RT6.1+

T cells in the peripheral lymphoid tissues (Gre-iner et al., 1986), although the RT6.1 gene isstructurally intact (Crisá et al., 1990). Preven-tion of IDDM in DP-BB/Wor rats by RT6+ Tcell engraftment, and induction of IDDM inDR-BB/Wor rats by RT6+ T cell depletion (Gre-

iner et al., 1987), led to the hypothesis thatIDDM outcome is determined by a balancebetween RT6− autoreactive T cells and RT6+

regulatory T cells (Mordes et al., 1987; Greineret al., 1988). Both lines of BB/Wor rats are alsosusceptible to autoimmune lymphocytic thy-roiditis, which is not discussed further in thisunit. Table 15.3.1 compares the salient featuresof IDDM in DP- and DR-BB/Wor rats.

Efficient induction of IDDM in both linesrequires both CD4+ and CD8+ cells (Metroz-Dayer et al., 1990; Edouard et al., 1993; Whalenet al., 1994). Genes for interferon γ (IFN-γ) andinterleukin 12 (IL-12) are expressed in BB/Worrat islets of both lines before and during IDDMonset, with little or no IL-4 or IL-10 observed(Zipris et al., 1996). The balance betweenautoreactive and regulatory T cells in DR-BB/Wor rats may therefore reflect a balancebetween TH1 and TH2 cell populations. Theinciting autoantigen(s) for T cells have notbeen identified, but the onset of hyperglyce-mia is preceded by the appearance of autoan-tibodies directed against a variety of tissueantigens (antigens expressed on the surfaceof islet cells, insulin, thyroid colloid, endo-thelial cells, smooth muscle, and gastric pa-rietal cells). Thymic epithelial cell defectshave been described in BB rats (Rozing et al.,1989; Doukas et al., 1994), and it has beenhypothesized that these defects may result infaulty intrathymic selection processes andsubsequent release of autoreactive T cellsfrom BB rat thymus.

Adoptive transfer of IDDM into histocom-patible WAG nude recipients can be achievedwith a diabetogenic inoculum administered atan appropriate cell dose. Fresh T cells fromacutely diabetic DP-BB/Wor rats fail to adop-tively transfer insulitis or diabetes, even at adose of 1 × 108 spleen cells. IDDM transfer ispossible if DP-BB/Wor spleen cells are acti-


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vated in vitro with concanavalin A prior totransfer (McKeever et al., 1990). The ineffec-tiveness of fresh DP-BB spleen cells in theadoptive transfer model is most likely due tothe short life span of peripheral T cells inthese animals. In contrast, IDDM transfer byDR-BB rat spleen cells, lymph node cells, orthymocytes does not require in vitro T cellactivation.

The use of animal models to study the patho-genesis of human IDDM provides numerousbenefits, but certain caveats apply (Rossini etal., 1995). It is not certain how accurately BBrats or nonobese diabetic (NOD) mice reflectthe pathogenesis of human IDDM. BB rats andNOD mice differ in that many treatment mo-dalities that prevent diabetes in NOD mice do

not prevent disease in BB rats. The efficacy ofthese modalities in preventing human IDDM isunknown.

Critical Parameters andTroubleshooting

Inbreeding in the various dispersed coloniesof BB rats worldwide has resulted in geneticpolymorphisms and significant differences inautoimmune characteristics among rats fromthe different colonies. For example, the diabe-tes-resistant BB-DR/Ed rat is lymphopenic, yetnone of the animals develop IDDM (Joseph etal., 1993). For studies not intended to addressidiosyncratic or strain-specific characteristics,the authors recommend use of BB/Wor ratsfrom the NIH-sponsored colony in Worcester,

Table 15.3.1 Characteristics of DP- and DR-BB/Wor Ratsa,b

Characteristic DP-BB/Wor DR-BB/Wor

IDDM Spontaneous Inducible

IDDM-inducing agents NA Anti-αRT6.1 MAb plus poly(IC);Anti-αRT6.1 MAb plus KRVinfection; KRV alone; poly(IC)alone

Prevention RT6+ T cell reconstitution,LCMV infection,immunosuppressive, andimmunomodulatory agents

NA

Frequency of IDDM 60%-80% in non-VAFhousing; >80% in VAF housing

30% with poly(IC) or KRV alone;>90% with anti-RT6 MAb pluspoly(IC); >90% with anti-RT6MAb plus KRV

Time of onset of IDDM 55-110 days of age (mean80 days)

14-21 days after treatment begins

Immunocompetence Lymphopenic, deficient inRT6+ and CD8+ T cells;indolent graft rejection;defective T cell response tomitogen

Normal T cell phenotype;immunocompetent

Adoptive transfer ofIDDM

Spleen and lymph node cells,requires mitogen activation

Spleen and lymph node cells donot require mitogen activation;thymocytes require treatment ofall recipients with anti-αRT6.1MAb and VAF-housed recipientswith poly(IC)

Diabetogenic effectorcells

CD4+ RT6.1− andCD8+ RT6.1−

CD4+ RT6.1− andCD8+ RT6.1−

Associated autoimmunity Thyroiditis Procedures that induce IDDMalso induce thyroiditis

aBased on Crisá et al. (1991) and Rossini et al. (1995).bAbbreviations: DP, diabetes prone; DR, diabetes resistant; IDDM, insulin-dependent diabetes mellitus; KRV, Kilhamrat virus; MAb, monoclonal antibody; NA, not applicable; poly(IC), polyinosinic/polycytidylic acid; VAF, viral antigenfree.


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Mass. These animals have been inbred for >50generations and are bred and maintained in avirus-antibody-free (VAF) barrier facility.

Selective brother-sister mating in the NIHcolony at Worcester has generated six sublinesof the DP-BB/Wor rat, designated BA, BB, BE,NA, NB, and PA. The cumulative frequency ofdiabetes averages 80% to 85% in all of thesesublines; mean age at onset of diabetes is 80days (range 55 to 110 days of age). The fre-quency of spontaneous diabetes is similar inmales and females. The sublines do vary withrespect to the incidence of spontaneous autoim-mune thyroiditis. There are two sublines of theDR-BB/Wor rat, designated WA and VB; bothare completely free of spontaneous diabeteswhen housed in a VAF facility. Male and femaleDR-BB rats are equally susceptible to sponta-neous or induced diabetes. BB rats certified asVAF can be ordered from Dennis Guberski,Department of Pathology, University of Mas-sachusetts Medical School, Worcester, Mass.,01655, Tel. (508) 856-3698, Fax (508) 856-1700. BB rats from this colony are seronegativefor the viruses and other pathogens listed inSupport Protocol 3. Information on other colo-nies of BB rats worldwide is available in Prinset al. (1991).

The most critical parameter for all of theprotocols described is proper control of theenvironment to which experimental rats are ex-posed. Environmental factors, especially dietand viruses, can contribute to either diseaseenhancement or inhibition (Crisá et al., 1992;Rossini et al., 1995). Not all such factors haveyet been identified or characterized. It is there-fore advantageous to attempt to control for asmany of these variables as possible. Obtain onlyVAF animals for experiments, and ensure thatanimal care staff strictly follow husbandry pro-cedures as outlined in the care of immunocom-promised animals (UNIT 1.2). Clidox disinfectant(see Basic Protocol 1) is of particular impor-tance because of its ability to destroy thoseviruses known to affect the frequency of IDDMin BB rats. The severe lymphopenia that affectsDP-BB rats renders them very susceptible toinfections, especially pneumonia caused by my-coplasma, which is lethal. DP-BB rats are alsosusceptible to environmental pathogens such aspinworms and to malignant lymphomas.

Anticipated ResultsThe cumulative frequency of IDDM and the

latency to disease onset in animals reported inthe various protocols and in Table 15.3.1 arethose observed in the University of Massachu-

setts Medical School barrier facility for breed-ing BB/Wor rats and in a VAF animal suitemaintained at the same institution. Outcomesmay vary if protocols are performed in non-VAF facilities or using BB rats obtained fromother colonies (Prins et al., 1991).

The authors have observed that >90% ofDR-BB/Wor rats that are serologically positivefor Kilham rat virus (KRV) and treated withanti-RT6.1 MAb alone, develop IDDM. Toachieve this rate using VAF DR-BB/Wor rats,the animals must be treated with both anti-RT6.1 MAb and poly(IC). Peripheral T cellsderived from either VAF or KRV-seropositiveacutely diabetic DR-BB rats can transfer IDDMinto WAG nu/nu recipients with comparableefficiency. Cotransfer of RT6+ T cells witheither diabetogenic inoculum will impair orprevent the adoptive transfer of IDDM.

Time ConsiderationsExperiments that use DP-BB rats and are

designed to examine frequency of IDDM typi-cally require 120 days to complete. Inductionof IDDM in DR-BB rats requires 14 to 21 daysof treatment. Adoptive transfer of diabetogenicT cells into nude recipients requires preparationof acutely diabetic DR-BB rat donors (14 to 21days after initiation of treatment). Latency toonset in the adoptive transfer model is a func-tion of cell dose, and can be achieved as earlyas 18 days after cell transfer. In all protocols,weights and urine glucose levels are measuredbeginning 4 to 5 days before IDDM onset isexpected and continuing three times weeklyuntil the end of the experiment.

Preparation of donor cells for transfusionand their injection into 5 to 10 recipient DP-BBrats (Basic Protocol 2) or WAG nu/nu rats(Alternate Protocol 2) requires 3 to 5 hr of anexperienced technician’s time. Treatment to in-duce diabetes in DR-BB rats requires intraperi-toneal injections of animals 5 consecutive daysper week for 2 to 3 weeks. The time requiredwill vary according to the number of animalsbeing treated.

Time requirements for screening animals atrisk for diabetes depend on the number ofexperimental animals that need to be evaluatedand on the rigor with which special proceduresto ensure VAF status of animal rooms are ob-served. Once gowned, a skilled technician canscreen 20 rats for weight and urinary glucoselevels in 1 hr, with an additional 1 to 1.5 hrrequired to obtain blood samples from 20 ratsfor confirmation of a diagnosis of IDDM.


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Literature CitedBurstein, D., Mordes, J.P., Greiner, D.L., Stein, D.,

Nakamura, N., Handler, E.S., and Rossini, A.A.1989. Prevention of diabetes in the BB/Wor ratby a single transfusion of spleen cells: Parame-ters that affect the degree of protection. Diabetes38:24-30.

Crisá, L., Greiner, D.L., Mordes, J.P., Handler, E.S.,Angelillo, M., Nakamura, N., and Rossini, A.A.1990. Biochemical studies of RT6 alloantigensin BB/Wor and normal rats: Evidence for intactbut unexpressed RT6a structural gene in diabe-tes-prone BB rats. Diabetes 39:1279-1288.

Crisá, L., Mordes, J.P., and Rossini, A.A. 1992.Autoimmune diabetes mellitus in the BB rat.Diabetes Metab.Rev. 8:9-37.

Doukas, J., Mordes, J.P., Swymer, C., Niedzwiecki,D., Mason, R., Rozing, J., Rossini, A.A., andGreiner, D.L. 1994. Thymic epithelial defectsand predisposition to autoimmune disease in BBrats. Am. J. Path. 145:1517-1525.

Dyrberg, T., Schwimmbeck, P.L., and Oldstone,M.B.A. 1988. Inhibition of diabetes in BB ratsby virus infection. J. Clin. Invest. 81:928-931.

Edouard, P., Hiserodt, J.C., Plamondon, C., andPoussier, P. 1993. CD8+ T cells are required foradoptive transfer of the BB rat diabetic syn-drome. Diabetes 42:390-397.

Greiner, D.L., Handler, E.S., Nakano, K., Mordes,J.P., and Rossini, A.A. 1986. Absence of the RT-6T cell subset in diabetes-prone BB/W rats. J.Immunol. 136:148-151.

Greiner, D.L., Mordes, J.P., Handler, E.S., Angelillo,M., Nakamura, N., and Rossini, A.A. 1987. De-pletion of RT6.1+ T lymphocytes induces diabe-tes in resistant BioBreeding/Worcester (BB/W)rats. J. Exp. Med. 166:461-475.

Greiner, D.L., Mordes, J.P., Angelillo, M., Handler,E.S., Mojcik, C.F., Nakamura, N., and Rossini,A.A. 1988. Role of regulatory RT6+ T-cells in thepathogenesis of diabetes mellitus in BB/Worrats. In Frontiers in Diabetes Research: Lessonsfrom Animal Diabetes II (E. Shafir and A.E.Reynolds, eds.) pp.58-67. John Libby, London.

Guberski, D.L., Thomas, V.A., Shek, W.R., Like,A.A., Handler, E.S., Rossini, A.A., Wallace, J.E.,and Welsh, R.M. 1991. Induction of type 1 dia-betes by Kilham’s rat virus in diabetes resistantBB/Wor rats. Science 254:1010-1013.

Guberski, D.L., Manzi, S.M., and Like, A.A. 1992.Correction of lymphopenia by injection of dia-betes-resistant splenocytes prevents diabetes andimproves breeding efficiency in diabetes-proneBB/Wor rats. Contemp. Top. Lab. Animal Sci.31:13.

Jacoby, R.O. and Ball-Goodrich, L.J. 1995. Parv-ovirus infections of mice and rats. Semin. Virol.6:329-337.

Joseph, S., Diamond, A.G., Smith, W., Baird, J.D.,and Butcher, G.W. 1993. BB-DR/Edinburgh: A

lymphopenic, nondiabetic subline of BB rats.Immunology 78:318-328.

McKeever, U., Mordes, J.P., Greiner, D.L., Appel,M.C., Rozing, J., Handler, E.S., and Rossini,A.A. 1990. Adoptive transfer of autoimmunediabetes and thyroiditis to athymic rats. Proc.Natl. Acad. Sci. U.S.A. 87:7718-7722.

Metroz-Dayer, M.-D., Mouland, A., Brideau, C.,Duhamel, D., and Poussier, P. 1990. Adoptivetransfer of diabetes in BB rats induced by CD4T lymphocytes. Diabetes 39:928-932.

Mordes, J.P., Desemone, J., and Rossini, A.A. 1987.The BB rat. Diabetes Metab.Rev. 3:725-750.

Nakhooda, A.F., Like, A.A., Chappel, C.I., Murray,F.T., and Marliss, E.B. 1977. The spontaneouslydiabetic Wistar rat. Metabolic and morphologicstudies. Diabetes 26:100-112.

Prins, J.-B., Herberg, L., Den Bieman, M., and VanZutphen, B.F.M. 1991. Genetic characterizationand interrelationship of inbred lines of diabetes-prone and not diabetes-prone BB rats. In Fron-tiers in Diabetes Research: Lessons from AnimalDiabetes III (E. Shafrir, ed.) pp. 19-24. Smith-Gordon, London.

Rozing, J., Coolen, C., Tielen, F.J., Weegenaar, J.,Schuurman, H.-J., Greiner, D.L., and Rossini,A.A. 1989. Defects in the thymic epithelialstroma of diabetes-prone BB rats. Thymus140:125-135.

Rossini, A.A., Mordes, J.P., Handler, E.S., and Gre-iner, D.L. 1995. Human autoimmune diabetesmellitus: Lessons from BB rats and NOD mice—Caveat emptor. Clin. Immunol. Immunopathol.74:2-9.

Thomas, V.A., Woda, B.A., Handler, E.S., Greiner,D.L., Mordes, J.P., and Rossini, A.A. 1991. Ex-posure to viral pathogens alters the expression ofdiabetes in BB/Wor rats. Diabetes 40:255-258.

Whalen, B.J., Greiner, D.L., Mordes, J.P., and Ross-ini, A.A. 1994. Adoptive transfer of autoimmunediabetes mellitus to athymic rats: Synergy ofCD4+ and CD8+ T cells and prevention by RT6+

T cells. 1994. J. Autoimmunity 7:819-831.

Whalen, B.J., Rossini, A.A., Mordes, J.P., and Gre-iner, D.L. 1995. DR-BB rat thymus containsthymocyte populations predisposed to autoreac-tivity. Diabetes 44:963-967.

Zipris, D., Greiner, D.L., Malkani, S., Whalen, B.,Mordes, J.P., and Rossini, A.A. 1996. Cytokinegene expression in islets and thyroids of BB rats.Interferon-γ and IL-12p40 mRNA increase withage in both diabetic and insulin-treated nondia-betic BB rats. J. Immunol. 156:1315-1321.

Contributed by Barbara J. Whalen, John P. Mordes, and Aldo A. RossiniUniversity of Massachusetts Medical SchoolWorcester, Massachusetts


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