Method of stimulating immune response

A method is disclosed for stimulating a mammal's or avian's immune response, particularly immune-compromised mammals, by administration of IGF-I, alone or in combination with growth hormone. Preferably, the IGF-I is native-sequence, mature human IGF-I.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a method of stimulating immune response in 
mammals or avian, including increasing antibody response to antigens in 
patients with depressed immune systems. 
2. Description of Related Art 
Insulin-like growth factor I (IGF-I) is a polypeptide naturally occurring 
in human body fluids, for example, blood and human cerebral spinal fluid. 
Most tissues, and especially the liver, produce IGF-I together with 
specific IGF-binding proteins. IGF-I production is under the dominant 
stimulatory influence of growth hormone (GH), and some of the IGF-I 
binding proteins are also increased by GH. See Tanner et al., Acta 
Endocrinol., 84:681-696 (1977); Uthne et al., J. Clin. Endocrinol. Metab., 
39:548-554 (1974)). IGF-I has been isolated from human serum and produced 
recombinantly. See, e.g., EP 123,228 and 128,733. 
Human growth hormone (hGH) is a single-chain polypeptide consisting of 191 
amino acids (molecular weight 21,500). Disulfide bonds link positions 53 
and 165 and positions 182 and 189. Niall, Nature, New Biology. 230:90 
(1971). hGH is a potent anabolic agent, especially due to retention of 
nitrogen, phosphorus, potassium, and calcium. Treatment of 
hypophysectomized rats with GH can restore at least a portion of the 
growth rate of the rats. Moore et al., Endocrinology, 122:2920-2926 
(1988). Among its most striking effects in hypopituitary (GH-deficient) 
subjects is accelerated linear growth of bone growth plate cartilage 
resulting in increased stature. Kaplan, Growth Disorders in Children and 
Adolescents (Springfield, Ill.: Charles C. Thomas, 1964). 
It has been reported that, especially in women after menopause, GH 
secretion declines with age. Millard et al., Neurobiol. Aging, 229-235 
(1990); Takahashi et al., Neuroendocrinology, 46:137-142 (1987). See also 
Rudman et al., J. Clin. Invest., 67:1361-1369 (1981) and Blackman, 
Endocrinology and Aging, 16:981 (1987). Moreover, a report exists that 
some of the manifestations of aging, including decreased lean body mass, 
expansion of adipose-tissue mass, and the thinning of the skin, can be 
reduced by GH treatment three times a week. See, e.g., Rudman et al., N. 
Eng. J. Med., 323:1-6 (1990) and the accompanying article in the same 
journal issue by Dr. Vance (pp. 52-54). 
The levels of IGF-I are reported to be reduced by half in 20-month old rats 
compared to 6-month old rats. Takahashi and Meiters, Proc. Soc. Exp. Biol. 
Med., 186:229-233 (1987). See also Florini and Roberts, J. Gerontol., 
35:23-30 (1980); Florini et et al., Mech. Aging Dev., 15:165-176 (1981); 
Chatelain et al., Pediatrie, 44:303-308 (1989); Florini et al., J. 
Gerontol., 40:2-7 (1985); Hall and Sara, Clinics in Endocrin, and Metab., 
15:629 (1984); Baxter, Advances in Clinical Chemistry, 25:49 (1986); 
Clemmons and Underwood, Clinics in Endocrin. and Metab., 28:629 (1986); 
Hintz, Advances in Pediatrics, 28:293 (Year Book Medical Publishers, Inc., 
1981); Johanson and Blizzard, The Johns Hopkins Medical Journal, 
149:115-117 (1981), the latter five references describing low IGF-I levels 
in aged men. The Hintz, Clemmons and Underwood, and Baxter references are 
general reviews on IGF-I. 
Furthermore, it was found that among human diploid fibroblasts capable of 
cycling in aging cultures in vitro, there were few changes in the 
regulation of the growth fraction by platelet-derived growth factor (PDGF) 
and epidermal growth factor (EGF), but a greatly increased dependence on 
IGF-I for regulation of the rate of entry into S phase. Chen and 
Rabinovitch, J. Cell. Physiol., 144:18-25 (1990). The authors conclude 
that the slower growth of the dividing population of cells in aging 
cultures may be related to a requirement for IGF-I at levels that are 
greatly above those usually supplied. This may be due to overproduction of 
the IGF-I binding protein, IGFBP-3, and, therefore, a reduction in IGF-I 
availability to its receptor. Goldstein et al., "Cellular and Molecular 
Applications to Biology of Aging", AFCR Meeting abstract, Seattle, May 
4-5, 1991. 
Various biological activities of IGF-I in other than aged mammals have been 
identified. For example, IGF-I is reported to lower blood glucose levels 
in humans. Guler et al , N. Engl. J. Med., 317:137-140 (1987). 
Additionally, IGF-I promotes growth in several metabolic conditions 
characterized by low IGF-I levels, such as hypophysectomized rats 
[Skottner et al., J. Endocr., 112:123-132 (1987)], diabetic rats 
[Scheiwiller et at., Nature, 323:169-171 (1986)], and dwarf rats.[Skottner 
et al., Endocrinology, 124:2519-2526 (1989)]. The kidney weight of 
hypophysectomized rats increases substantially upon prolonged infusions of 
IGF-I subcutaneously. Guler et al., Proceedings of the 1st European 
Congress of Endocrinology, 103: abstract 12-390 (Copenhagen, 1987). The 
kidneys of Snell dwarf mice and dwarf rats behaved similarly. van 
Buul-Offers eg al., Pediatr. Res., 20:825-827 (1986); Skottner et al. 
Endocrinology, supra. An additional use for IGF-I is to improve glomerular 
filtration and renal plasma flow. Guler et al., Proc. Natl. Acad. Sci. 
USA, 86: 2868-2872 (1989). The anabolic effect of IGF-I in rapidly growing 
neonatal rats was demonstrated in vivo. Philipps et al., Pediatric Res., 
23:298 (1988). In underfed, stressed, ill, or diseased animals, IGF-I 
levels are well known to be depressed. 
GH and IGF-I have been linked with immunoregulatory properties. The immune 
response results from interaction of antigens (foreign or non-self 
moieties) with host cells (lymphocytes) bearing specific receptors on the 
surface membrane for these antigens. Lymphocytes are grouped into two 
major classes, T-cells and B-cells. 
T-cells originate from the thymus where they mature and differentiate from 
bone-marrow-derived cells. The mature T-cells leave the thymus gland to 
continuously circulate from blood to lymph nodes and spleen and back to 
blood. T-cells are further subdivided into three major subsets: T-helper 
cells, T-suppressor cells, and T-cytolytic cells. T-helper cells "help" 
other cells: B-cells to secrete antibody, cytotoxic cells to become 
functional, and macrophages to become activated. This population of 
T-cells bears the CD.sub.4 surface marker that is used to identify this 
subset in tissue and blood. 
T-cytolytic cells are responsible for killing target cells such as virally 
infected cells, tumor cells, and allografts. Suppressor T-cells act to 
limit and terminate the immune response. The cytolytic and suppressor 
T-cell populations are identified by the CD.sub.8 surface marker. 
The B-cells, or antibody-forming cells, also derive from immature 
precursors found in the bone marrow. When mature, the B-cells migrate to 
all lymphoid organs except the thymus. B-cells interact with antigens by 
way of antibody molecules bound to their plasma membranes that act as 
receptor proteins. This surface immunoglobulin is used as a marker to 
identify B-cells in tissue and blood. Following interaction with antigen 
and T-helper cells, the B-cells differentiate into antibody-forming cells 
called plasma cells. These plasma cells secrete antibody into the 
extracellular matrix. The antibody diffuses into capillaries and 
circulates via normal blood flow. Thus, the serum immunoqlobulin level 
reflects the cellular dynamics of the immune response. 
In many states, children are required to be immunized routinely against 
such diseases as diphtheria, pertussis, and typhoid (DPT), as well as 
measles, tetanus, mumps, polio, and rubella, by administering vaccines. 
The B-cell reaction to vaccine is the production of appropriate 
immunoglobulins, which are intended to confer immunity against the 
disease. Generally, a particular B-cell will be differentiated to produce 
one particular type of antibody, and such production is caused by the 
presence in the body of one particular type of antigen. Hence, when an 
animal or person has been exposed to a number of different antigens, the 
animal or human will have a number of different B-cells that can produce 
its particular immunoglobulins when the appropriate antigen is present. 
In some situations, the immune response to antigen is insufficient to 
confer immunity. That is, a quantity of immunoglobulins is generated (or a 
number of B-cells are potentiated) that is insufficient to confer 
effective immunity. 
It has been known since 1967 that a connection exists between the anterior 
pituitary and the immune system, and specifically with GH. Two groups of 
investigators concluded from their studies that GH controls the growth of 
lymphoid tissue. Pierpaoli and Sorkin, Nature, 215:834 (1967); Baroni, 
Experientia, 23:282 (1967). Subsequently, immunologic function was 
restored in the pituitary dwarf mouse by a combination of bovine 
somatotropic hormone and thyroxin. Baroni et al., Immunol., 17:303-314 
(1969). 
In a sex-linked dwarf chicken strain, bovine GH treatment resulted in 
enhanced antibody responses and bursal growth while thyroxine treatment 
stimulated thymus growth. Marsh et al., Proc. Soc. Exp. Biol. Med., 
175:351-360 (1984). However, neither treatment altered immune function in 
the autosomal dwarf chicken. Bovine GH therapy alone partially restored 
immunologic function in immunodeficient Weimaraner dogs. Roth et al., Ann. 
J. Vet. Res., 45:1151-1155 (1984). 
Mice with hereditary GH deficiency develop an impairment of the immune 
system associated with thymic atrophy, immunodeficiency, and wasting, 
resulting in a shortened life expectancy. Frabris et al., Clin. Exp. 
Immunol., 9:209-225 (1971). It has been shown that an age-associated 
decline in the plasma concentration of thymulin (a thymic hormone) occurs 
and that plasma thymulin concentration increases in bGH-treated 
middle-aged and old dogs. Goff et al., Clin. Exp. Immunol., 68:580-587 
(1987). The authors suggest that exogenous GH may be useful for restoring 
some immune functions in aged individuals. Further, administration of hGH 
to C.sub.57 /B1/6J mice was found to reverse the inhibitory effect of 
prednisolone on thymus and spleen cellularity and on natural killer 
activity; administration of hGH without prednisolone had no effect, 
although at higher doses it induced a decrease of thymic parameters and 
natural killer activity with no effect on spleen cellularity, and relative 
weights. Franco et al., Acta Endocrinologica, 123:339-344 (1990). 
It has also been shown that GH induces T-cell proliferation in the thymus. 
Murphy et al., FASEB Meeting Abstract, Atlanta, April 1991; Durum et al., 
FASEB Meeting Abstract, Atlanta, April 1991. For recent reviews on the 
immune effects of GH, see Kelley, "Growth Hormone in Immunobiology," in 
Psychoneuroimmunology II, 2nd Ed., B. Ader et al., eds., Acad. Press 1990, 
and Ammann, "Growth Hormone and Immunity," in Human Growth 
Hormone--Progress and Challenges, L. Underwood, ed., Marcel Dekker, Inc., 
New York, (1988), pp. 243-253; Weigent and Blalock, Prog. 
NeuroEndocrinImmunology, 231-241 (1990). It has been reported that the 
activity of all major immune cell types, including T-cells, B-cells, 
natural killer (NK) cells and macrophages, can be altered by GH. Kelly, 
Biochem. Pharmacol., 38:705 (1989). 
One report states that locally generated IGF-I mediates GH action on 
T-lymphocytes through the type I IGF receptor. Geffner et al., J. Clin. 
Endocrin, and Metab., 71:464 (1990 ). Also, Franco et al., on p. 343, 
speculate that some of the effects of hGH on the immune system occur via 
IGF-I. Timsit et al., 73rd Annual Meeting, Endocrine Society, June 19-22, 
1991, abstract 1296, reports hGH and IGF I stimulate thymic hormone 
function. 
There have been data published documenting the ability of cells of the 
immune system to produce IGF-I-like molecules. These include activated 
alveolar macrophages [Rom et al., J. Clin. Invest., 82:1685 (1988)], human 
B-lymphocytes transformed with Epstein-Barr virus [Merimee et al., J. 
Clin. Endocrin. Metab., 69:978 (1989)], spleen and thymus tissues through 
detection of mRNA for IGF-I [Murphy et al., Endocrinology, 120:1279 
(1987)], and normal T-cells [Geffner et al., supra]. 
Data have also been presented suggesting that IGF-I produced locally in 
tissues such as the thymus or inflammatory sites might affect the growth 
and function of IGF-I-receptor-bearing T-lymphocytes. Tapson et al., J. 
Clin. Invest., 82:950-957 (1988). 
A statistically significant increase in thymus and spleen weight of 
hypophysectomized rats infused for 18 days with IGF-I was observed as 
compared to control or treatment with GH. Froesch et al., in Growth 
Hormone Basic and Clinical Aspects. eds. O. Isaksson et al., p. 321-326 
(1987). Also reported was an increased thymic tissue in young GH-deficient 
rats treated with IGF-I [Guler et al., Proc. Natl. Acad. Sci. USA, 
85:4889-4893 (1988)] and an increase in the spleen of dwarf rats [Skottner 
et al., Endocrinology, supra]. Others have shown repopulation of the 
atrophied thymus in diabetic rats using either IGF-I or insulin; however, 
when the rats were immunized with bovine serum albumin (BSA) and boosted, 
serum anti-BSA antibodies showed no effect of insulin or IGF-I on the 
antibody response despite large effects on thymic and splenic size. Binz 
et al., Proc. Natl. Acad. Sci. (USA). 87:3690-3694 (1990). IGF-I was 
reported to stimulate lymphocyte proliferation (Johnson et al., Endocrine 
Society 73rd Annual Meeting, abstract 1073, June 19-22, 1991). 
Furthermore, IGF-I was found to repopulate the bone marrow cavity with 
hematopoietic cells [Froesch et al., supra], stimulate erythropoiesis in 
hypophysectomized rats [Kurtz et al., Proc. Natl. Acad. Sci. (USA). 
85:7825-7829 (1988)], and enhance the maturation of morphologically 
recognizable granulocytic and erythroid progenitors in suspension cultures 
of marrow cells. Merchav et al., J. Clin. Invest., 81:791 (1988). 
At nanomolar concentrations, IGF-I is a growth-promoting factor for 
lymphocytes. Schimpff et al., Acta Endocrinol., 102:21-25 21-25 (1983). 
B-cells, but not T-cells, have recently be to possess receptors for IGF-I. 
Stuart et al., J. Clinical Endo. and Met., 72:1117-1122 (1991). Also, 
IGF-I, as a chemotactic for resting and activated T-cells, stimulates an 
increase in thymidine incorporation into resting and activated T-cells. 
Normal T-cell lines show augmentation of basal colony formation in 
response to IGF-I. Geffner et al., supra. It is also stated on p. 955 of 
Tapson et al., J. Clin. Invest., 82:950-957 (1988) that IGF-I produced 
locally in tissues such as the thymus or inflammatory sites might affect 
the growth and function of IGF-I receptor-bearing T lymphocytes. However, 
IGF-I is reported to suppress in a dose-dependent manner IL-2-induced 
proliferative responses and in vitro antibody responses of splenocytes. 
Hunt and Eardley, J. Immunol., 136:3994-3999 (1986). 
There is a need in the art to supply a reagent that will stimulate the 
immune system of a mammal or avian, whether the immune response is 
cell-mediated or antibody-mediated. There is a particular need for a 
reagent that will boost the antibody response of patients with compromised 
immune systems to antigens to which they are exposed. In view of the 
controversy in the art surrounding IGF-I, it is unclear what its effects 
would be in increasing immune function, as opposed to merely increasing 
size of organs involved in immune function such as the thymus and spleen, 
or in increasing the activity of T- or B-cells in vitro or in vivo. 
It is therefore an object of the present invention to stimulate the immune 
response of a mammal or avian. 
It is a particular object to increase production of immunoglobulins by 
increasing the number of immunoglobulin-producing cells and/or by 
increasing the amount of immunoglobulin produced by the individual 
immunoglobulin-producing cells in response to the predetermined immunogen. 
It is a more particular object to increase antibody responses in patients 
with severely hampered immune systems, such as patients who receive bone 
marrow transplants or in AIDS patients. 
These and other objects will be apparent to those of ordinary skill in the 
art. 
SUMMARY OF THE INVENTION 
Accordingly, the present invention provides a method for stimulating a 
mammal's or avian's immune system comprising administering to the mammal 
or avian an immune-stimulating effective amount of IGF-I. 
In a more particular aspect, the invention provides a method for increasing 
a mammal's or avian's antibody response to an immunogen comprising 
administering to the mammal or avian the immunogen and an effective amount 
of IGF-I. Preferably, this administration is concurrent and is followed by 
boosts of immunogen at shortened intervals relative to if no IGF-I is 
given. 
In another aspect, the invention provides co-administration of effective 
amounts of IGF-I and GH for stimulating the immune system. 
In still another aspect, a method is provided of increasing the amount of 
immunoglobulin produced by B-cells of a human or other mammalian subject 
in response to an immunogen, where said subject suffers from a condition 
in which insufficient immunoglobulin production occurs, comprising 
administering to the subject an effective amount of IGF-I, the amount 
being effective to increase the production of immunoglobulin. 
In a still further aspect, the invention provides a method of increasing 
the T-cell responsiveness in a human or other mammalian subject in 
response to an immunogen, where said subject suffers from a condition in 
which insufficient T-help or T-cytolytic activity occurs, comprising 
administering to the subject an effective amount of IGF-I, the amount 
being effective to increase the T-help or T-cytolytic activity. 
In yet another aspect, the invention provides a method of treating an 
immune-deficient mammal or avian comprising: 
(a) measuring the serum IGF-I level of the mammal; and 
(b) if the serum IGF-I level is below a normal level for that 
mammal or avian, administering to the mammal or avian an 
effective amount of IGF-I to restore immunity. 
While recent studies in whole animals mentioned above have shown that IGF-I 
can cause increased spleen and thymus weights in GH-deficient animals, 
these studies have not progressed beyond describing a gross change in 
thymus and spleen size or in cell number. Other manipulations of the size 
of the spleen and thymus have been shown not to be associated with an 
effect on function. Jardieu and Fraker, J. Immunol.. 124:2650-2655 (1980). 
Furthermore, the Binz et al. article cited above utilized a diabetic rat 
model where insulin and IGF-I would affect diabetes and therefore aid all 
tissues in the body, and IGF-I and insulin were found to have no 
functional effect on antibody titer. 
In view of this art, the present invention represents an unexpected finding 
that not only are the spleen and thymus weights increased upon 
administration of IGF-I, but also the function of the thymus, spleen, or 
lymph nodes, as indicated by increased splenocyte number, splenic T-cell 
population number, splenic B-cell number, and their responses to mitogens 
in vitro. The increase in B-cell number and responsiveness is now shown to 
translate to increased production of antibody by these cells in response 
to an antigen. This method would be useful in treating patients having 
compromised immune systems such as AIDS patients, in whom increased 
antibody response to antigens would ward off, or decrease the severity of, 
infectious diseases and in whom vaccines could be made more effective. 
Wherever IGF-I is used, it is reasonable to expect that IGF-II will 
similarly function.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A. Definitions 
As used herein, "stimulating an immune system" refers to increasing the 
immune function of a mammal or avian, whether the increase is due to 
antibody mediation or cell mediation, and whether the immune system is 
endogenous to the host treated with IGF-I or is transplanted from a donor 
to the host recipient given IGF-I (such as bone marrow transplants). For 
example, the stimulation may result from an increased number of splenic 
cells such as splenic lymphocyte number, splenic T-cell population number 
(T-cell, CD.sub.4 and CD.sub.8), or splenic B-cell number, or from an 
increased number of thymocytes. Other cells involved in the immune system 
response include natural killer cells, macrophages, and neutrophils. In 
addition, the stimulation may be due to an increase in antibody production 
in response to an immunogen. 
As used herein, the expressions "compromised immune system" and "condition 
in which insufficient immunoglobulin production occurs" signify the immune 
system of humans as well as animals that have a smaller antibody response 
to antigens than normal, whether because their spleen size is smaller than 
it should be, whether the spleen is only partially functional, whether 
drugs such as chemotherapeutic agents are suppressing the normal immune 
function, whether the animal is functionally IGF-I (or GH) deficient, or 
due to any other factor. Examples include aged patients, patients 
undergoing chemotherapy or radiation therapy, recovering from a major 
illness, or about to undergo surgery, patients with AIDS, patients with 
congenital and acquired B-cell deficiencies such as hypogammaglobulinemia, 
common varied agammaglobulinemia, and selective immunoglobulin 
deficiencies, e.g., IgA deficiency, patients infected with a virus such as 
rabies with an incubation time shorter than the immune response of the 
patient, and patients with hereditary disorders such as diGeorge syndrome. 
The mammals and avians potentially affected herein include mammals and 
avians of economic importance such as bovine, ovine, and porcine animals, 
as well as chickens and turkeys. The mammals may exhibit a splenic atrophy 
and subsequent loss in B-cell number and function The preferred mammal 
herein is a human. 
As used herein, "IGF-I" refers to insulin-like growth factor from any 
species, including bovine, ovine, porcine, equine, avian, and preferably 
human, in native-sequence or in variant form, and from any source, whether 
natural, synthetic, or recombinant. Preferred herein for animal use is 
that form of IGF-I from the particular species being treated, such as 
porcine IGF-I to treat pigs, ovine IGF-I to treat sheep, bovine IGF-I to 
treat cattle, etc. Preferred herein for human use is human 
native-sequence, mature IGF-I, more preferably without a N-terminal 
methionine, prepared, e.g., by the process described in EP 230,869 
published Aug. 5, 1987; EP 128,733 published Dec. 19, 1984; or EP 288,451 
published Oct. 26, 1988. More preferably, this native-sequence IGF-I is 
recombinantly produced and is available from Genentech, Inc., South San 
Francisco, Calif. for clinical investigations. Also preferred for use is 
IGF-I that has a specific activity greater than about 14,000 units/mg as 
determined by radioreceptor assay using placenta membranes, such as that 
available from KabiGen AB, Stockholm, Sweden. 
The most preferred IGF-I variants are those described in PCT WO 87/01038 
published Feb. 26, 1987 and in pCT WO 89/05822 published June 29, 1989, 
i.e., those wherein at least the glutamic acid residue is absent at 
position 3 from the N-terminus of the mature molecule or those having a 
deletion of up to five aming acids at the N-terminus. The most preferred 
variant has the first three aming acids from the N-terminus deleted 
(variously designated as brain IGF, tIGF-I, des(1-3)-IGF-I, or des-IGF-I). 
As used herein, "GH" refers to growth hormone from any species, including 
bovine, ovine, porcine, equine, avian, and preferably human (hGH), in 
native-sequence or in variant form, and from any source, whether natural, 
synthetic, or recombinant. This includes both Met-hGH [U.S. Pat. No. 
4,755,465 issued Jul. 5, 1988 and Goeddel et al., Nature. 282:544 (1979)], 
which is sold under the trademark PROTROPIN.RTM. by Genentech, Inc. and is 
identical to the natural polypeptide, with the exception of the presence 
of an N-terminal methionine residue, and recombinant hGH (rhGH), available 
to clinical and research investigators from Genentech, Inc. under the 
trademark Nutropin.RTM., and commercially available from Eli Lilly, that 
lacks this methionine residue and has an aming acid sequence identical to 
that of the natural hormone. See Gray et al., Biotechnology, 2:161 (1984). 
Both met-hGH and rhGH have equivalent potencies and pharmacokinetic 
values. Moore et al., supra. Another suitable hGH candidate is an hGH 
variant that is a placental form of GH with pure somatogenic and no 
lactogenic activity. U.S. Pat. No. 4,670,393 issued 2 June 1987. 
As used herein, the expression "increasing antibody response to an 
immunogen" refers to raising the serum immunoglobulin (IgG) titer of an 
animal in response to a boost of the antigen against which the IgG is 
directed. Indicators of increased antibody response include an increase in 
the production of antibodies to booster shots of immunogen, as well as an 
increase in the number of B-cells in the patient. The immunogen can be any 
that raise antibodies directed thereto, but preferably is a virus, 
including a vaccine, or a bacterium. The invention is particularly useful 
for those instances where the mammal or avian is infected with a virus 
that has an incubation time that is shorter than the immune response of 
the mammal or avian, such as, e.g., rabies. The IGF-I herein decreases the 
interval between primary and secondary immunizations or between secondary 
immunization and subsequent boosts of immunogen. 
As used herein, the expression "increasing the T-cell responsiveness to an 
immunogen" in a subject suffering from a condition in which insufficient 
T-help or T-cytolytic activity occurs refers to raising the level of 
T-helper and/or T-cytolytic cell activity of the mammal in response to an 
immunogen to which T-cells are responsive, including viral antigens, 
tumors, bacteria, etc. A subject with insufficient T-help or T-cytolytic 
activity is a mammal that has less than the normal number of T-helper 
and/or T-cytolytic cells (as determined, e.g., by CD.sub.4 /CD.sub.8 
markers) necessary to, for example, secrete antibodies, activate 
macrophages, and kill target cells such as virally infected or tumor 
cells. 
As used herein, the expression "restore immunity" in a mammal means to 
bring the level of immunity of the mammal back to normal, whether by 
restoring splenic or thymic cells or by increasing T-cell responsiveness 
or the amount of immunoglobulin produced by B-cells. 
B. Modes for Carrying Out the Invention 
For the various purposes of this invention, the IGF-I is directly 
administered to the mammal or avian by any suitable technique, including 
parenterally, and can be administered locally or systemically. The 
specific route of administration will depend, e.g., on the medical history 
of the patient, including any perceived or anticipated side effects using 
IGF-I. Examples of parenteral administration include subcutaneous, 
intramuscular, intravenous, intraarterial, and intraperitoneal 
administration. 
Most preferably, the administration is by continuous infusion (using, e.g., 
minipumps such as osmotic pumps), or by injection using, e.g., intravenous 
or subcutaneous means. Preferably, the administration is subcutaneous for 
IGF-I. The administration may also be as a single bolus or by slow-release 
depot formulation. Most preferably, the IGF-I is administered continuously 
by infusion, most preferably subcutaneously. 
In addition, the IGF-I is suitably administered together with any one or 
more of its binding proteins, for example, IGFBP-2, IGF-BP-4, or, most 
preferably, IGFBP-3, which is described in WO 89/09268 published Oct. 5, 
1989 and by Martin and Baxter, J. Biol. Chem., 261:8754-8760 (1986). This 
glycosylated protein is an acid-stable component of about 53 Kd on a 
non-reducing SDS-PAGE gel of a 125-150 Kd glycoprotein complex found in 
human plasma that carries most of the endogenous IGFs and is also 
regulated by GH. The IGF-I is also suitably coupled to a receptor or 
antibody or antibody fragment for administration. 
The IGF-I composition to be used in the therapy will be formulated and 
dosed in a fashion consistent with good medical practice, taking into 
account the clinical condition of the individual patient (especially the 
side effects of treatment with IGF-I alone), the site of delivery of the 
IGF-I composition, the method of administration, the scheduling of 
administration, and other factors known to practitioners. The "effective 
amount" of IGF-I for purposes herein (including an immune-stimulating 
effective amount) is thus determined by such considerations. 
As a general proposition, the total pharmaceutically effective amount of 
the IGF-I administered parenterally per dose will be in the range of about 
1 .mu.g/kg/day to 10 mg/kg/day of patient body weight, although, as noted 
above, this will be subject to therapeutic discretion. More preferably, 
this dose is at least 0.01 mg/kg/day, and most preferably for humans 
between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, 
the IGF-I is typically administered at a dose rate of about 1 
.mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4 injections per day 
or by continuous subcutaneous infusions, for example, using a mini-pump. 
An intravenous bag solution may also be employed. The key factor in 
selecting an appropriate dose is the result obtained, as measured by 
increases in antibody production, increases in splenocyte or thymocyte 
number, increases in splenic B-cells, etc. 
A course of IGF-I treatment to affect the immune system appears to be 
optimal if continued longer than a certain minimum number of days, 7 days 
in the case of the mice. The length of treatment needed to observe changes 
and the interval following treatment for responses to occur appear to vary 
depending on the desired effect. 
The IGF-I is also suitably administered by sustained-release systems. 
Suitable examples of sustained-release compositions include semi-permeable 
polymer matrices in the form of shaped articles, e.g., films, or 
microcapsules. Sustained-release matrices include polylactides (U.S. Pat. 
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and 
gamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers, 547-556 (1983)), 
poly(2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. 
Res., 15:167-277 (1981), and R. Langer, Chem. Tech., 12:98-105 (1982)), 
ethylene vinyl acetate (R. Langer et al., id.) or 
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release IGF-I 
compositions also include liposomally entrapped IGF-I. Liposomes 
containing IGF-I are prepared by methods known per se: DE 3,218,121; 
Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82:3688-3692 (1985); Hwang 
et al., Proc. Natl. Acad. Sci. U.S.A., 77:4030-4034 (1980); EP 52,322; EP 
36,676; EP 88,046; Ep 143,949; Ep 142,641; Japanese Pat. Appln. 83-118008; 
U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the 
liposomes are of the small (about 200-800 Angstroms) unilamellar type in 
which the lipid content is greater than about 30 mol. percent cholesterol, 
the selected proportion being adjusted for the optimal IGF-I therapy. 
For parenteral administration, in one embodiment, the IGF-I is formulated 
generally by mixing it at the desired degree of purity, in a unit dosage 
injectable form (solution, suspension, or emulsion), with a 
pharmaceutically acceptable carrier, i.e., one that is non-toxic to 
recipients at the dosages and concentrations employed and is compatible 
with other ingredients of the formulation. For example, the formulation 
preferably does not include oxidizing agents and other compounds that are 
known to be deleterious to polypeptides. 
Generally, the formulations are prepared by contacting the IGF-I uniformly 
intimately with liquid carriers or finely divided solid carriers or both. 
Then, if necessary, the product is shaped into the desired formulation. 
Preferably the carrier is a parenteral carrier, more preferably a solution 
that is isotonic with the blood of the recipient. Examples of such carrier 
vehicles include water, saline, Ringer's solution, and dextrose solution. 
Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful 
herein, as well as liposomes. 
The carrier suitably contains minor amounts of additives such as substances 
that enhance isotonicity and chemical stability. Such materials are 
non-toxic to recipients at the dosages and concentrations employed, and 
include buffers such as phosphate, citrate, succinate, acetic acid, and 
other organic acids or their salts; antioxidants such as ascorbic acid; 
low molecular weight (less than about ten residues) polypeptides, e.g., 
polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or 
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino 
acids, such as glycine, glutamic acid, aspartic acid, or arginine; 
monosaccharides, disaccharides, and other carbohydrates including 
cellulose or its derivatives, glucose, mannose, or dextrins; chelating 
agents such as EDTA; sugar alcohols such as mannitol or sorbitol; 
counterions such as sodium; and/or nonionic surfactants such as 
polysorbates, poloxamers, or PEG. 
The IGF-I is typically formulated in such vehicles at a concentration of 
about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 
8. Full-length IGF-I is generally stable at a pH of no more than about 6; 
des(1-3)-IGF-I is stable at about 3.2 to 5. It will be understood that use 
of certain of the foregoing excipients, carriers, or stabilizers will 
result in the formation of IGF-I salts. 
In addition, the IGF-I, preferably the full-length IGF-I, is suitably 
formulated in a suitable carrier vehicle to form a pharmaceutical 
composition that does not contain cells. In one embodiment, the buffer 
used for formulation will depend on whether the composition will be 
employed immediately upon mixing or stored for later use. If employed 
immediately, the full-length IGF-I can be formulated in mannitol, glycine, 
and phosphate, pH 7.4. If this mixture is to be stored, it is formulated 
in a buffer at a pH of about 6, such as citrate, with a surfactant that 
increases the solubility of the GH at this pH, such as 0.1% polysorbate 20 
or poloxamer 188. The final preparation may be a stable liquid or 
lyophilized solid. 
IGF-I to be used for therapeutic administration must be sterile. Sterility 
is readily accomplished by filtration through sterile filtration membranes 
(e.g., 0.2 micron membranes). 
Therapeutic IGF-I compositions generally are placed into a container having 
a sterile access port, for example, an intravenous solution bag or vial 
having a stopper pierceable by a hypodermic injection needle. 
IGF-I ordinarily will be stored in unit or multi-dose containers, for 
example, sealed ampoules or vials, as an aqueous solution or as a 
lyophilized formulation for reconstitution. As an example of a lyophilized 
formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) 
aqueous lGF-1 solution, and the resulting mixture is lyophilized. The 
infusion solution is prepared by reconstituting the lyophilized IGF-I 
using bacteriostatic Water-for-Injection. 
Also, GH may be combined with the IGF-I for this purpose, in a dose and 
using a suitable administration as is used for IGF-I above. It is noted 
that hGH is stable at a higher pH than IGF-I, e.g., 7.4-7.8. When GH is 
administered, it is suitably administered together with one or more of its 
binding proteins. A well characterized such binding protein is the 
high-affinity growth hormone binding protein (GHBP) constituting the 
extracellular domain of the GH receptor that circulates in blood and 
functions as a GHBP in several species [Ymer and Herington, Mol. Cell. 
Endocrino., 41:153 (1985); Smith and Talamantes, Endocrinology, 
123:1489-1494 (1988); Emtner and Roos, Acta Endocringlogica (Copenh.), 
122:296-302 (1990)], including man. Baumann et al., J. Clin. Endocringl. 
Metab., 62:134-141 (1986); EP 366,710 published May 9, 1990; Herington et 
al., J. Clin Invest., 77:1817-1823 (1986); Leung et al., Nature, 
330:537-543 (1987). A second BP with lower affinity for GH has also been 
described that appears to be structurally unrelated to the GH receptor. 
Baumann and Shaw, J. Clin. Endocringl. Metab., 70:680-686 (1990). 
The doses of both GH and IGF-I can be less if used together than if IGF-I 
is administered alone. It is noted that practitioners devising doses of 
both IGF-I and GH should take into account the known side effects of 
treatment with these hormones. For hGH the side effects include sodium 
retention and expansion of extracellular volume [Ikkos et al., Acta 
Endocringl. (Copenhagen), 32:341-361 (1959); Biglieri et al., J. Clin. 
Endocrinol. Metab., 21:361-370 (1961)], as well as hyperinsulinemia and 
hyperglycemia. The major apparent side effect of IGF-I is hypoglycemia. 
Guler et al., Proc. Natl. Acad. Sci. USA. 1989, supra. 
Preferably, the IGF-I is administered in conjunction with (i.e., at the 
same time as or after) a vaccine, such as an AIDS vaccine (for example, a 
gp120 or gp160 vaccine or a cocktail of gp receptor-based vaccines), 
either during initial immunization or during a boost of the vaccine, to 
ensure increased antibody response. Most preferably, the IGF-I is given at 
the time of each boost. The use of IGF-I with vaccine will increase the 
effectiveness of the vaccine, particularly in those patients who have 
compromised immune systems. 
It is another embodiment of this invention to diagnose immune-deficient 
mammals to determine if they have low serum IGF-I levels that could cause 
their malady and that could be reversed by treatment with IGF-I. Such 
human patients might include those who are aged, underfed, malnourished, 
or ill. Diagnosing the serum IGF-I level of such immune-deficient patients 
and restoring IGF-I blood concentrations in those patients with 
lower-than-normal serum IGF-I levels by administering an amount of IGF-I 
effective for that purpose would restore immunity in the patient. 
Diagnosing IGF-I levels in a patient can be accomplished by any standard 
technique, but is typically done by subjecting a blood sample to an ELISA 
or RIA test using anti-IGF-I antibodies such as described in Furlanetto et 
al., J. Clin. Invest., 60:648-657 (1977); Bala and Bhaumick, J. Clin. 
Endocrin. and Metabol., 49:770-777 (1979); and Zapf et al., J. Clin. 
Invest., 68:1321-1330 (1981). 
The invention will be more fully understood by reference to the following 
examples. They should not, however, be construed as limiting the scope of 
the invention. All literature and patent citations are expressly 
incorporated by reference. 
EXAMPLE I 
Evaluation of Organ Weights, B- and T-Cell Numbers, And Response to 
Mitogenic Stimulation 
Recombinant human IGF-I [available commercially from KabiGen AB, Stockholm, 
Sweden (specific activity&gt;14,000 U/mg by radioreceptor assay using 
placental membranes) or available for clinical investigations from 
Genentech, Inc., South San Francisco]was employed in all the IGF-I 
experiments detailed in the examples. The IGF-I was dissolved at 5 mg/ml 
in 10 mM citrate buffer and 126 mM NaCl, pH 6.0. 
This IGF-I was administered to three species, i.e., rat, rabbit, and mouse, 
to observe its effects on spleen and thymus weight. Dose-response studies 
were performed in the mouse and rat, and IGF-I was given to the rabbit 
with similar effects. In addition, B- and T-cell numbers and responses to 
mitogenic stimulation were evaluated in the mice. 
I. Rats 
Two animal models of GH deficiency and therefore IGF-I deficiency were used 
to demonstrate the effect of IGF-I on spleen and thymic weight and size. A 
third model of GH and IGF-I deficiency is the aged animal. Aged 
(18-month-old) rats were used to demonstrate the effect of IGF-I on spleen 
and thymic size, cellulants architecture, and in vitro response to 
mitogens. Also, adult ovariectomized rats, with normal serum IGF-I 
concentrations, were used to demonstrate the effect of IGF-I on spleen and 
thymus in an animal that was not IGF-I deficient. 
A. Dwarf Rats 
Female dwarf rats (Simonsen Labs, Gilroy, CA) (100-140 g) were dosed by 
subcutaneous (sc) infusion from osmotic mini-pumps for one week with 
IGF-I. FIG. 1 provides a dose-response graph for IGF-I on spleen size in 
these dwarf rats. Clearly, IGF-I is a very potent stimulant to splenic 
growth in the dwarf rat. 
B. Hypophysectomized Rats 
Female hypophysectomized rats (Taconic Farms, Germantown, NY), weighing 
85-105 g, were implanted sc with osmotic mini-pumps that delivered IGF-I 
and des-IGF-I [PCT WO 87/01038 published Feb. 26, 1987 and in pCT WO 
89/05822 published Jun. 29, 1989] over one week. The treatment with IGF-I 
and des-IGF-I shows a greatly enhanced growth response of the spleen and 
the thymus, as indicated in FIGS. 2A and 2B, respectively. This growth is 
greater than that of the whole body, as when the weight of the spleen or 
thymus is expressed per gram of body weight, there is still a very 
significant growth of the spleen and thymus. Both IGF-I and des-IGF-I have 
this activity, with des-IGF-I being significantly more potent than IGF-1 
in this regard. 
C. Adult Female Rats 
Adult female rats were ovariectomized. Thirty days later when the rats 
weighed 300 g they were implanted with osmotic minipumps (Alza, Palo Alto, 
2ML2) containing IGF-I (delivering 1.33 or 4 mg/kg/day of IGF-I) or 
excipient. At sacrifice 14 days after minipump implantation, the spleens 
were dissected and weighed (the thymus was not dissected in this 
experiment). 
FIG. 3 shows the dose-response graph for IGF-I in this rat model. It can be 
seen that even in a pituitary intact animal with normal endogenous growth 
hormone and IGF-I it was possible to demonstrate a large effect of 
exogenous IGF-I on body weight (an average gain of 45 g) and spleen 
weight. Even when the spleen weight was expressed as a percentage of body 
weight, very significant growth of the spleen could be demonstrated 
(***p&lt;0.001 vs. excipient, **p&lt;0.01 vs. excipient). 
Therefore, in the rat, IGF-I could be seen to affect the growth of tissues 
with immune functions in GH- and IGF-I-deficient animals (immune-deficient 
animals) and in animals with normal GH and IGF-I concentrations 
(immune-competent animals). 
D. Aged Rats 
In two separate in vivo studies, IGF-I, GH, or IGF-I plus GH were 
administered for 14 days to aged 18-month-old rats to determine whether 
IGF-I could induce functional changes in spleen and thymus in this model 
of thymic regression. 
(i) Design 
Male Fischer 344 rats of 18 months of age and 400-500 g were purchased from 
Harlan Sprague Dawley (HSD). These rats were bred by HSD for the NIH 
Institute for Aging and are the standard rat model used in aging studies. 
In Experiment One, 7 rats/group were employed, and in Experiment Two, 8 
rats/group. Young F344 rats (5-8 weeks old), which were housed identically 
as experimental rats, were used as positive controls. The treatment groups 
were: (1) excipient pumps, excipient injections, (2) IGF-I pumps, 
excipient injections, (3) IGF-I pumps, GH injections, (4) excipient pumps, 
GH injections, and (5) young rats. 
The IGF-I was loaded into two minipumps so that 1.150 mg/rat/day of IGF-I 
or 0.8 mg/kg/day of des-IGF-I was delivered sc as a continuous infusion. 
The rhGH (Nutropin.RTM. brand, Genentech, Inc. formulated at 2 mg/ml in 18 
mg/ml mannitol, 0.68 mg/ml glycine, and 5 mM phosphate, pH 7.4) or bGH 
(Monsanto) was given as a daily sc injection of 1 mg/rat/day. The 
excipient pump groups received identical pumps filled with the excipient 
for IGF-I (10 mM citrate buffer and 126 mM NaCl, pH 6.0), hereinafter 
called "IGF-I excipient." The treatments continued for 14 days. The 
animals no receiving GH were injected (0.1 ml) with hGH vehicle each day. 
At sacrifice, a blood sample was taken, and the liver, kidneys, heart, 
spleen, and thymus were removed, blotted dry, and immediately weighed. The 
spleen and thymus were immediately placed in buffer and then cells were 
obtained by digestion or physical rupture. The cells were counted and then 
plated out at uniform density. The thymic cells were cultured with IL-1 (2 
U/ml) and phytohemaglutinin (PHA) (5 .mu.g/ml) and thymidine incorporation 
was measured as described by Maizel et al., J. Exp. Med., 153:470-476 
(1981). The spleens were similarly treated and two tests of function were 
performed. 
(ii) Results 
(a) Experiment One 
Full-length IGF-I and rhGH were employed in this experiment. FIG. 4 shows 
the body weight gain. After 14 days control rats had not gained weight. 
GH-treated rats gained 9.6.+-.11.4 g, IGF-I-treated rats gained 
34.5.+-.9.4 g, and IGF-I- and GH-treated rats gained 45.5.+-.9.9 g. The 
response to IGF-I was clearly large, and the response to GH plus IGF-I 
appeared to be additive. IGF-I at the doses used was markedly anabolic. A 
very dramatic effect of IGF-I treatment was the large fall in blood urea 
nitrogen (BUN) levels from 20.7.+-.2.4 mg/dL in controls to 13.8.+-.1.8 
mg/dL after IGF-I treatment; hGH had no effect. A lowered BUN indicates an 
anabolic metabolic state. The body weight gain data, the increased organ 
weights, the lowered BUN, and the lowered blood enzyme levels all indicate 
that IGF-I was producing an anabolic state where protein synthesis was 
predominant over protein breakdown. The effect of IGF-I was clearly 
greater than that of hGH. 
There was a clear effect of IGF-I on all the organ weights. Liver increased 
by 6.6%, kidneys by 16.6%, heart by 18.5%, thymus by 27.0%, and spleen by 
80.8%. All the responses were statistically significant. The only effect 
of hGH was to reduce liver weight significantly by 8.8%. Combined GH and 
IGF-I treatment did not reduce the magnitude of the effect of IGF-I on 
these organs, with one exception. Spleen weight was reduced for the IGF-I 
plus GH treatment compared to the weight of the spleen in the IGF-I alone 
group. 
Total IGF-I levels were increased by IGF-I administration with or without 
concurrent hGH treatment. By itself, hGH did not significantly elevate 
blood total IGF-I levels. 
The cells from the harvested organs were dispersed and their response to 
mitogens was measured. Table I shows some of the data for the thymus and 
spleen. The wet weight of the thymus was increased by IGF-I but not by 
hGH. Normal, young, 60-day-old Fischer rats were run as positive controls. 
TABLE I 
______________________________________ 
Cell Number in Spleen (.times.10.sup.8) and Thymus (.times.10.sup.7) 
No. Thymic 
Group No. Spleen Cells 
Cells 
______________________________________ 
Young Rats 2.81 .+-. 0.30 
4.43 .+-. 
0.79*** 
Old Rats Excipient 
2.72 .+-. 0.68 
0.19 .+-. 
0.15 
Old Rats IGF-1 3.58 .+-. 0.86 
0.96 .+-. 
0.66** 
Old Rats IGF-1 + GH 
3.27 .+-. 1.47 
0.82 .+-. 
0.27*** 
Old Rats GH 2.50 .+-. 0.51 
0.36 .+-. 
0.28* 
______________________________________ 
Values are Means and Standard deviations. 
(Significances: *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001 vs Excipient) 
None of the thymi from the untreated old rats yielded sufficient cells to 
allow full analysis in tissue culture. In contrast, 8 of the 13 rats 
treated with IGF-I or IGF-I plus GH did yield sufficient viable thymic 
cells. IGF-I treatment for 14 days caused a remarkable 5-fold increase in 
the number of thymic cells, although the thymus of the younger rats still 
contained substantially more cells. 
Growth hormone tended to increase the number of thymic cells, but the 
effect (a doubling of the mean number) was not statistically significant. 
IGF-I plus hGH was also an effective way to increase thymic cell number. 
In contrast, the number of cells in the spleen was not significantly 
increased by IGF-I or GH treatment, although the mean values of the 
IGF-I-treated groups were higher. Therefore, IGF-I could increase the wet 
weight of the thymus and also the number of cells capable of being 
harvested. Then, any functional effect of the increased tissue mass and 
cell number was tested in vitro by measuring the responses of the 
dispersed thymocytes to mitogens, as shown in Table II below. 
For both the PHA and IL-1 responses and their combination, the tissue from 
the old rats showed a tendency toward increased activity with IGF-I alone 
compared to that from the younger animals, although this effect was not 
statistically significant. There was no additive effect of the IGF-I plus 
GH combination on the number of cells harvested. It was therefore 
surprising that IGF-I plus GH had the largest and most significant effect 
on all measures of thymic function. Compared to the responses of the 
younger tissue, the PHA response for IGF-I plus GH was increased 3.7-fold 
and for the PHA plus IL-1 combination the response was increased 4-fold. 
These data show that an increased mass of thymic tissue can be produced in 
an aged animal using IGF-I, and after the relatively short period of only 
14 days of IGF-I treatment. There are previous studies in similarly aged 
rats that show that both GH and prolactin can increase the size and some 
aspects of thymic 
TABLE II 
__________________________________________________________________________ 
Thymic cells from young and old F344 rats. Untreated old rats all had 
insufficient thymic cells to run the assays. 
Treatment 
Cell No. 
PHA IL-1 PHA + IL-1 
__________________________________________________________________________ 
Young Rat 
4.96 1764 1360 3349 
Young Rat 
4.80 1790 989 3836 
Young Rat 
3.52 2112 1462 3629 
Mean 1888 .+-. 
193 1270 .+-. 
249 3604 .+-. 
244 
Old Rats 
IGF-1 0.37 3078 672 11273 
IGF-1 1.72 3524 1028 3724 
IGF-1 1.68 3032 854 6532 
IGF-1 1.20 1523 929 -- 
Mean 2789 .+-. 
872* 870 .+-. 
150** 
7176 .+-. 
3815* 
Old Rats 
IGF-1 + GH 
0.92 10436 1536 18990 
IGF-1 + GH 
1.06 5120 2836 17446 
IGF-1 + GH 
1.12 7432 2316 13429 
IGF-1 + GH 
0.78 5095 1796 7865 
Mean 7020 .+-. 
2526## 
2121 .+-. 
576# 
14432 + 
4966## 
Old Rats 
GH 0.72 2005 581 4371 
GH 0.82 11263 1780 27021 
Mean -- -- -- -- 
__________________________________________________________________________ 
Values are mean c.p.m. from individual animals, the group means are based 
on these values 
Comparisons (#IGF1 + GH vs Young; *IGF1 vs IGF1 + GH) 
(Significances: *p &lt; 0.05, **p &lt; 0.01, #p &lt; 0.05, ##p &lt; 0.01) function. 
Kelley, in Psychoneuroimmunology II, 2nd Ed., B. Ader et al., eds, 1990, 
supra. 
It has also now been shown that the increased thymic tissue produced by 
IGF-I is functional tissue, in that it can respond to mitogens. There were 
four times as many thymic cells in the young rats, but the cells from 
IGF-I-treated old rats had an in vitro activity that was improved up to 
4-fold. Therefore, according to the functional tests used, the thymus of 
the older rats was essentially restored to that of a much younger animal. 
In the thymus the effect of aging appeared to have been reversed. 
(b) Experiment Two 
In a second set of 18-month-old rats, a similar experiment was performed, 
except that bGH and des-IGF-I were employed. Also tested was the activity 
of des-IGF-I and whether the relatively poor effect of hGH in the first 
study was due to hGH antibodies (GH is very antigenic in the rat, bGH much 
less so). 
The results are shown in Table III. The weight gains with des-IGF-I seemed 
less than in the first study, but were still superior to the response to 
bGH. The kidney and spleen showed large responses to des-IGF-I, and no 
significant response to GH. In general, des-IGF-I returned the blood cell 
counts toward those in the younger animals, with the combination of 
des-IGF-I and bGH being the most affective treatment. des-IGF-I tended to 
increase the white blood cell (WBC) and the lymphocyte number when 
combined with bGH. This change is similar in amount to that seen in 
Example IV, in man. 
The results of thymic weight, cell number, and percentage of cells that 
were PNA (peanut agglutinin) positive are shown in Table IV. It can be 
seen that thymus weight was increased at sacrifice in the 
des-IGF-I-treated rats. This experiment was designed to test the origin 
and type of increased cell number in the thymus. This discrimination of 
the origin and type of cells was achieved by FACS analysis (described 
further below) using PNA as the specific marker for true thymocytes. PNA 
positive thymocytes are believed to be young precursor cells for T-cells. 
TABLE III 
__________________________________________________________________________ 
Blood Counts: Aged rats treated with des-IGF-1 and GH; cf. Young Rats 
Lympcte RBC Platelet 
Group WBC No Hematocrit 
no MCV No. 
__________________________________________________________________________ 
a Control Old 
7.36 .+-. 1.42 
4.32 .+-. 0.75 
38.0 .+-. 1.8 
7.59 .+-. 0.49 
50.1 .+-. 1.1 
676 .+-. 29 
b des-IGF-1 
8.12 .+-. 0.76 
4.23 .+-. 0.41 
37.8 .+-. 1.8 
7.29 .+-. 0.38 
51.8 .+-. 0.5 
726 .+-. 69 
c bGH 6.97 .+-. 0.96 
4.15 .+-. 0.76 
37.4 .+-. 1.3 
7.39 .+-. 0.32 
50.6 .+-. 0.5 
795 .+-. 46 
d des + bGH 
8.93 .+-. 1.90 
4.80 .+-. 1.16 
37.6 .+-. 1.2 
7.01 .+-. 0.22 
53.9 .+-. 1.6 
783 .+-. 98 
e Young Rats 
8.92 .+-. 1.24 
6.40 .+-. 0.81 
37.5 .+-. 0.9 
6.53 .+-. 0.14 
57.4 .+-. 1.0 
897 .+-. 68 
__________________________________________________________________________ 
Means and Standard Deviations n = 7 & 8, except for group (e) where (n = 
4). 
TABLE IV 
______________________________________ 
Thymus Cell Counts: Aged F344 rats treated 
with des-IFG-1 and bGH; cf. Young Rats 
Thymus Wt Cell No. PNA+ 
Group (mg) (.times.10.sup.6) 
(%) 
______________________________________ 
a Excipient Old 
80 .+-. 35 0.66 .+-. 
0.2 24 .+-. 
12 
b des-IGF-1 (0.64 
117 .+-. 
27* 3.27 .+-. 
2.1** 
72 .+-. 
14*** 
c bGH (1.0) 
66 .+-. 17 1.30 .+-. 
0.6 37 .+-. 
18 
d des + bGH 
144 .+-. 
39** 2.79 .+-. 
1.5** 
69 .+-. 
23*** 
e Young Rats 
338 .+-. 
30*** 2.85 .+-. 
0.8** 
94 .+-. 
2*** 
______________________________________ 
Means and Standard Deviations n = 7 & 8, except for group e (n = 4). 
The young rats had 5-fold more thymic cells than the old rats. The number 
of cells in the thymus was increased about 4.5-fold using des-IGF-I alone 
or in combination with bGH. By itself, bGH increased cell number only 
two-fold. These responses confirm the observations in Experiment One. The 
percentage of the cells that were PNA positive was unexpected. The young 
control rats had 95% PNA positive cells, and the aged rats only 25% 
positive cells. 
Des-IGF-I by itself in these old rats increased the percentage PNA positive 
cells to 72% of the cells. A similar number (69%) was seen for the 
des-IGF-I plus bGH group. bGH by itself did not significantly affect the 
percentage PNA positive cells. This indicates that "real" thymic 
repopulation was being regenerated in the old animals, composed of 
precursor cells for T-cells. 
Therefore, des-IGF-I produced a very dramatic effect by returning both the 
number of cells and the percentage that were PNA positive essentially to 
normal. IGF-I appears to have a marked effect on the rejuvenation of the 
thymus in an aged rat. At t sacrifice in Experiment Two in the aged rats, 
half the thymus was placed in 10% formalin and histological sections were 
prepared. The general morphology of the thymus was assessed by a 
veterinary pathologist as being characterized by (1) no significant 
lesions (the young control animals), or (2) involution (normal for the 
aged animals), or (3) showing evidence of lymphocytic hyperplasia. In 
addition, the amount of lymphocytic cellularity within the thymus was 
graded for all the animals, as this seemed to be the cell component that 
was different between the groups. 
Using this scheme characteristic, thymic involution was seen in the 
excipient and the GH-treated groups. However, there was clear evidence of 
lymphocytic hyperplasia and the restoration of the thymic architecture in 
the groups that received des-IGF-I and des-IGF-I plus bGH. The increase in 
the lymphocytic cellularity in the rats treated with des-IGF-I was easily 
distinguishable. Scoring the slides for the degree of involution and the 
amount of lymphocytic hyperplasia confirmed that involution was 
significantly reversed by des-IGF-I (p&lt;0.01, Fisher's test) and that the 
amount of lymphocytic hyperplasia was greatly increased by des-IGF-I 
(p&lt;0.001). Therefore, histological examination of the thymus confirmed 
that IGF-I can rejuvenate the thymus of an aged animal, even where thymic 
involution has already occurred. 
II. Rabbits 
Male New Zealand White rabbits 2.0-2.5 kg were anesthetized and renal 
damage was induced by clamping both renal arteries for 120 minutes. At 
clamping, either one Alzet osmotic pump (Alza Corporation, Palo Alto, CA, 
Model 2ML-1) containing 2 ml of 3.3 mg des-IGF-I/ml acetic acid (1 00 mM, 
pH 4.5), or 2 Alzet osmotic pumps containing 2 ml each of 5.0 mg IGF-I/ml 
(in sodium chloride/sodium acetate buffer, pH 6.0) were placed in the 
abdominal cavity. The pumps delivered either 0.364 mg of des-IGF-I/kg/day 
or 1.18 mg IGF-I/kg/day for 7 days. Control animals received 
excipient-filled pumps. The animals were sacrificed at day 7 and the 
thymus and spleen were dissected. 
After seven-day treatment with IGF-I the average wet weight of the thymus 
in IGF-I-treated rabbits (n=6) was 4.7.+-.0.44 g, nearly twice as large as 
those of the control animals (2.7.+-.0.58 g, n=4, p=0.023). When thymus 
size was expressed as a percentage of rabbit body weight the statistical 
significance of the effect increased (p=0.014). 
After seven-day treatment with des-IGF-I, the average wet weight of the 
spleen in treated rabbits (n=8, 2.43.+-.0.44 g) was more than twice as 
large as that of the control rabbits (n=7, 1.17.+-.0.21 g, p=0.028). 
III. Mice 
The above studies using rats and rabbits established that IGF-I could cause 
profound changes in the immune system. The mouse was next used as a model 
system, as in this species immune cell markers and assays are better 
characterized and were readily available. Furthermore, it was desired to 
establish in the mouse if the effects on thymus and spleen size, cell 
number, and in vitro responses to mitogens were translated into a real 
functionally enhanced activity of the immune system. 
Since it was shown that in aged rats IGF-I had remarkable activity in 
restoring the architecture and cytology of the thymus to that of a young 
animal and that the cells produced showed enhanced mitogenic response, 
aged mice were chosen as the model, in this case retired breeder male 
mice, which are a model of accelerated aging. The effect of IGF-I as an 
anabolic agent as well as an effector of immune tissue growth and function 
was studied in the adult aged mice. In addition, the effect of hGH and a 
combination of IGF-I and hGH on cell number and mitogenic stimulation was 
evaluated. 
A. Design 
1. Protocol 
The following studies used retired breeder BALB/c mice 9 months old or 
older and weighing approximately 25 to 35 g (Harlan Sprague Dawley, San 
Diego, Calif.). Animals were housed in single cages and given food (Purina 
Rodent Chow 5010, St. Louis, Mo.) and water, ad libitum. All animals were 
weighed before being grouped into treatment groups (based on their body 
weight) using a randomization program. Animals were identified with 
stainless steel ear tags and were acclimated for at least one week. 
IGF-I was administered by sc-implanted osmotic minipump (for 7-day studies, 
Alzet Model 2001, pump rate approximately 1 .mu.l/hr.; for 14-day studies, 
two Alzet Model 2002 minipumps, pump rate approximately 0.5 .mu.l/hr; 
Alza, Palo Alto, Calif.). The pumps were loaded with solution per the 
manufacturer's instructions, and the filled pumps were then incubated in 
sterile saline overnight in the refrigerator. 
The pumps were filled with either the IGF-I excipient or the desired 
concentration of IGF-I (5 mg/ml formulated as described above), i.e., 7.5, 
30, or 120 .mu.g IGF-I/day/7 days for 6 animals per group for the first 
seven-day treatment study and 120 .mu.g IGF-I/day/7 days for 5 animals per 
group for the second seven-day treatment study and the 14-day treatment 
study. 
For hGH treatments, rhGH (Nutropin.RTM. brand) was administered by itself 
in an amount of 9.6, 48, or 240 .mu.g hGH/day/14 days via two Alzet Model 
2002 osmotic minipumps (0.5 .mu.l/hr/14 days) implanted sc to 5 animals 
per group, or by itself via 240 .mu.g hGH for 14 days via sc injection, 5 
animals/group. 
For combination studies of IGF-I and GH, IGF-I was administered in a dose 
of 120 .mu.g by two Alzet 2002 minipumps and GH was administered by daily 
sc 240-.mu.g injections into 5 animals/group. 
The mice were anesthetized with an ip injection of approximately 0.4 ml of 
avertin (2,2,2-tribromoethanol and tert-amyl alcohol in phosphate buffered 
saline (PBS)). The dorsal scapular region was then clipped of hair and a 
small incision was made. A close hemostat was then inserted into the 
incision and pushed posteriorly. A minipump was then inserted into the 
pocket and the incision was closed with stainless steel wound clips, and a 
sc injection of 7500 U of penicillin was given. Animals were inspected 
daily and their body weights recorded. 
Animals were sacrificed at various times following minipump placement, a 
large blood sample was taken, and the thymus, spleen, heart, liver, 
kidney, and mandibular and mesenteric lymph nodes from each treatment 
group were removed aseptically and weighed. The spleen, thymus, and lymph 
nodes were placed on ice in tissue culture media in separate vials for 
further assays. All data are expressed as the mean.+-.standard deviation, 
with comparisons being made by one-way analysis of variance with follow-up 
comparisons being made using Duncan's Range Test. 
3. Cell Preparation 
The lymph nodes, spleen and thymus were dispersed using sintered glass 
slides to form single cell suspensions. The cells were then washed, in 
Eagle's minimal essential medium (MEM, Gibco, Grand Island, N.Y.) 
containing 10% fetal bovine serum (FBS) (Gibco), penicillin (100 
units/ml), 100 .mu.g/ml streptomycin (Gibco), and 200/mM glutamine, and 
resuspended at 5.times.10.sup.6 viable cells/ml as determined by trypan 
blue dye exclusion. 
4. Mitogen Stimulation 
Lipopolysaccharide (LPS - E. coli 055:B5) was obtained from Difco 
Laboratories (Detroit, Michigan). Pokeweed mitogen (PWM) and Concanavalin 
A (Con A) were obtained from Sigma (St. Louis, Mo.). The response to each 
mitogen was assayed in triplicate at the following concentrations: LPS 
(100, 10, 1 .mu.g/ml), PWM (10, 5, 2.5 .mu.g/ml), Con A (10, 5, 2.5 
.mu.g/ml). Two hundred microliters of cells (2.5.times.10.sup.6 /ml) 
containing the appropriate dilution of mitogen were cultured in 
flat-bottom microtiter plates (Falcon Plastics, Oxnard, Calif.) in Hepes 
(0.5M)-NaHCO:-buffered (0.24% w/v) MEM containing 10% FBS and supplements 
as described above. Cultures were incubated at 37.degree. C. in 10% 
CO.sub.2. 
After 72 hours, the cultures were pulsed with 1 mCi of methyl .sup.3 
H-thymidine. Twelve hours later, the cultures were harvested onto glass 
fiber filters using a multiple sample harvester. Discs were dried and 
placed in 3 ml of scintillation fluid. The amount of .sup.3 H-thymidine 
incorporated into DNA was measured using a Beckman scintillation counter. 
Only optimal responses to mitogens, which were the same for all treatment 
groups, were reported. 
5. FACS Analysis 
Lymphocyte cell suspensions prepared as described were adjusted to 
1.times.10.sup.6 cells/ml in PBS containing 0.1% BSA and 10 mM sodium 
azide. Two-hundred-microliter aliquots of the cell suspensions were 
incubated for one hour at 4.degree. C. with 5 .mu.l of the appropriate 
dilution of monoclonal rat anti-mouse FITC conjugate anti-thy-1, 
anti-L3T4, or anti-Lyt-2 (Caltag, S. San Francisco, Calif.) to stain the 
T-cell populations. B-cells in these suspensions were stained using 
FITC-conjugated F(ab').sub.2 polyclonal goat anti-mouse Ig (M,G,A 
specific) (Becton Dickinson, Mountainview, Calif.). Following three washes 
with cold medium, cells were analyzed for degree of fluorescence intensity 
using a FACS 440 (Becton Dickinson, Sunnyvale, Calif.). Fluorescence 
parameters were collected using a log amplifier after gating on the 
combination of forward and perpendicular light scatter. Fluorescence data 
was expressed as percentage of fluorescent cells compared to non-relevant 
mab of identical isotypes. Fluorescence was measured as mean fluorescence 
intensity of the fluorescent cells as expressed as mean channel number 
plotted on a log scale. 
B. 7 and 14 Day Studies 
The purpose of these studies was to establish if IGF-I was anabolic in the 
intact normal mouse and if at such anabolic doses IGF-I affected thymic 
and splenic weight, cellularity, cell type, and responsiveness in vitro to 
mitogens. Five or six mice per group were used in these studies. On the 
basis of the doses known to be effective in the rat, it was decided to 
deliver IGF-I by continuous sc infusion at 140, 46, and 15 .mu.g/mouse/day 
(approximately 4, 1.33, and 0.44 mg/kg/day). 
C. Results 
1. Effect of 7-Day Treatment 
There was a dose-related effect on body weight gain over the 7 days 
(excipient 0.75.+-.0.75 g, low dose 0.86.+-.0.63 g, medium dose 
1.31.+-.1.03 g, and high dose 3.42.+-.1.24 g), with the high-dose response 
being highly statistically significant compared to all other groups 
(p&lt;0.001). In the repeat experiment with the high-dose IGF-I a similar 
weight gain (3.55.+-.0.54 g) occurred that again was statistically greater 
(p&lt;0.001) than the gain of the excipient-treated group. 
IGF-I caused significant growth of the spleen and the thymus after 7 days 
of treatment with IGF-I. In the first experiment there was a clear 
dose-related effect of IGF-I on the spleen (excipient 105.+-.14, low dose 
124.+-.21; medium dose 145.+-.58; high dose 193.+-.23 mg; excipient vs. 
high-dose IGF-I, p&lt;0.001). In the repeat experiment, the spleen weight 
again increased (excipient 103.+-.18, high dose 206.+-.68 mg, p=0.01). 
Thymus weight was unchanged in the first experiment; this was probably due 
to the thymus being dissected differently by different dissectors. In the 
repeat experiment, one dissector uniformly removed the thymus, and 
significant thymic growth was detected (excipient, 15.2.+-.1.3; high dose 
26.2.+-.6.4 mg, p=0.006). 
The observed increase in spleen weight following seven-day treatment with 1 
40 .mu.g IGF-I/day was due in part to an increase in lymphocyte number. 
Viable lymphocytes, as determined by trypan blue exclusion, increased from 
2.times.10.sup.8 to 5.times.10.sup.8 cells/spleen following 7-day 
treatment with IGF-I (FIG. 5). This increase in cell number appeared to be 
due to an increase in both B- and T-cells. When B- and T-cell numbers were 
quantitated by FACS analyses of SIg+and Thy 1+cells, respectively, B-cell 
number increased 3 fold (1.3.times.10.sup.8 excipient vs. 
3.5.times.10.sup.8 IGF-I), while T-cell number was also increased compared 
to controls (0.7.times.10.sup.7 excipients vs. 1.1.times.10.sup.7 IGF-I). 
See FIG. 5. 
The observed increase in thymic weight correlated with an increase in Thy 
1+thymocytes (1.times.10.sup.7 excipient vs. 2.4.times.10.sup.7 IGF-I). 
See FIG. 6. These data suggest that IGF-I has a potent mitogenic effect on 
lymphocyte subpopulations. 
In contrast to the dramatic increase in lymphocyte number induced by IGF-I, 
the response of splenic lymphocytes to stimulation by LPS (B-cells) and 
Con A (T-cells) was decreased compared to controls, while the response to 
PWM was equivalent for both groups of mice. See FIG. 7. This depressed 
mitogenic response suggests a lack of functional maturity in the 
lymphocyte population following short-term (7-day) IGF-I treatment. 
Therefore, in the 7-day experiment, lymphocyte number was increased, but 
mitogenic response was depressed. 
2. Effect of 14-Day Treatment 
Next it was determined if a longer exposure to IGF-I was required to effect 
lymphocyte function than was required to effect lymphocyte number. 
Therefore, treatment was extended to 14 days using the high dose of IGF-I 
(140 .mu.g/mouse/day). Furthermore, since hGH is thought to act in part by 
inducing IGF-I production, the effects of hGH vs. IGF-I on lymphocyte 
responses were compared. 
There was a significant weight gain after 14 days of treatment with IGF-I 
(excipient 1.49.+-.0.46; high dose 3.87.+-.0.45 g, p&lt;0.001). Additionally 
there was significant splenic growth (excipient 96.+-.12; high dose 
163.+-.9, p&lt;0.001), and significant thymic growth (excipient 18.2.+-.4.6: 
high dose 33.8.times.10.6, p=0.017). It can be seen that the thymus and 
spleen reached similar weights after 7 or 1 4 days of treatment. 
As seen in the 7-day experiment, the spleen cell number nearly doubled 
(1.3.times.10.sup.8 vs. 2.4.times.10.sup.8) compared to controls using 
IGF-I treatment (FIG. 8). While there was an increase in T-cell number in 
the IGF-I-treated mice, the only statistically significant increase was 
seen in the CD. population (3.1.times.10.sup.7 vs. 4.9.times.10.sup.7) 
(FIG. 8), suggesting that CD4+cells may be preferentially increased by 
this treatment regime. As seen in the previous experiment, IGF-I treatment 
resulted in substantial increases in B-cell number. IGF-I also showed an 
increase in T-cell number in the thymus when treatment was carried out for 
14 days. See FIG. 9. 
In contrast to the decreased response seen at 7 days, following 14 days of 
IGF-I treatment the mitogenic response of splenocytes from IGF-I-treated 
mice was significantly elevated compared to controls (FIG. 1 0). These 
data suggest that short-term administration of IGF-I results in 
significant increases in lymphocyte number, but additional time is 
required to see alterations in lymphocyte responsiveness. 
3. Effect of Combination After 14-Day Treatment 
a. Simultaneous Treatment 
Since hGH and IGF-I had different effects on lymphocyte populations, in the 
next series of experiments the effects of hGH administered simultaneously 
with IGF-I were examined. Whether alone or in combination with sc-injected 
hGH, IGF-I treatment produced increases in total lymphocyte number in the 
spleen, which again appeared to be due primarily to an increase in B-cell 
number (FIG. 11). The combination of IGF-I and hGH did have a pronounced 
effect on thymocyte number over IGF-I or hGH treatment alone (FIG. 12). 
It is expected that the preferred route of combination therapy would be 
administration of continuously infused IGF-I and hGH. 
b. Sequential Treatment 
When GH (at 280 .mu.g/day) was administered first for 14 days followed by 
administration by IGF-I (at 140 .mu.g/day) for 14 days, no effect of IGF-I 
was seen. 
4. Long-Term Effects of 14-Day Treatment 
To determine the long-lasting effects of IGF-I, hGH and the combination, 
lymphocyte populations from control and treated animals were examined 7 
and 21 days after 14-day treatment with hGH, IGF-I, or the combination of 
IGF-I and hGH. 
Seven days post-treatment the IGF-I- and IGF-I- plus hGH-treated mice had 
significantly elevated splenocyte numbers compared to either control, or 
hGH-treated mice (FIG. 13). A statistical increase in B-cell number was 
observed in both IGF-I-treated groups. The increase in T-cell number was 
significant in the IGF-I only group, but not in the combination of hGH 
plus IGF-I group. Furthermore, both CD+and CDs+T-cell populations were 
elevated in this group compared to controls. As was the case with 14-day 
treatment, both groups of IGF-I-treated mice had elevated thymocyte 
numbers compared to hGH-treated or control mice (FIG. 14). In addition, 
IGF-I, alone or in combination with hGH, produced an increase in 
peripheral lymph node cell numbers (FIG. 16). No alteration in node T cell 
number or CD.sub.4 :CD.sub.8 ratios was observed following these treatment 
regimes. 
Unlike the enhanced proliferative response to mitogens seen at 14 days of 
treatment, the mitogenic responses of the IGF-I-treated mice had returned 
to control values by 7 days after treatment (FIG. 15). The largest 
mitogenic responses were seen in the hGH- plus IGF-I-treated group 
compared to controls, but these differences were not statistically 
significant. 
By 21 days after treatment, all four groups of mice had equivalent 
splenocyte (FIG. 17) and thymocyte (FIG. 18) numbers. Thus, 21 days 
appears to be sufficient to restore the normal cell number and phenotypic 
ratios following IGF-I treatment. 
However, by 21 days after treatment, both the LPS and Con A responses of 
the hGH- plus IGF-I-treated group were statistically elevated compared to 
controls (FIG. 19). Similarly, the responses to all three mitogens were 
elevated in the IGF-I only group. These results suggest that IGF-I has an 
early and late acting effect on lymphocyte responses, while the 
combination of IGF-I and hGH appears to require some time to effect 
lymphocyte responsiveness. sc-Injected hGH alone failed to have a 
statistically significant effect on mitogen responses at any time point 
examined. 
EXAMPLE II 
Response to Antigen in Secondary Immunization 
The purpose of this experiment was to evaluate the immune function in male 
mice (retired breeders) immunized with dinitrophenyl-ovalbumin and treated 
with IGF-I. Previous experiments indicated that 14 days of continuous 
IGF-I administration to retired male breeder mice increased the body 
weight, spleen, and thymus organ weights. It was shown that the increase 
in spleen weight was attributable to an increase in B-cell number and an 
increase in mitogen responsiveness. It was also shown that increased 
T-cell numbers in the thymus could be generated and that these cells were 
also more responsive to mitogens. These data indicated that if IGF-I 
caused the antibody-producing B-cells and the helper T-cells to be greater 
in number and more responsive to mitogens, then IGF-I might be able to 
give a greater antibody response to an antigen. 
I. Protocol 
Forty-eight hours after arrival, all animals received a single ip injection 
(100 .mu.l) of dinitrophenyl-ovalbumin mixed with alum (DNPOA). (The 
dinitrophenyl group is a hapten that elicits a B-cell-dependent response, 
and the ovalbumin is a carrier that elicits a T-cell dependent response.) 
The DNPOA was mixed before use by adding 50 .mu.l of DNPOA (1 mg/ml) to 
2.45 ml of sterile PBS, pH 7.0 and 2.50 ml of aluminum hydroxide 
absorptive gel (Rehsorptar.TM. brand, sold by Armor Pharmaceutical Col, 
IL, 20 mg/ml). The DNPOA was mixed for approximately 30 minutes prior to 
injection. The day of DNPOA immunization is designated as Day 0. 
At Day:19, ten animals were grouped by body weight into two groups. (One 
animal was found dead on day 9.). Nineteen miniosmotic pumps (Alzet Corp., 
Palo Alto, Calif.) model 2002 (0.5 .mu.l/hr, 14 days) were filled with 
IGF-I excipient or IGF-I as described in Example I and placed in sterile 
saline solution overnight at 4.degree. C. 
At Day 20, five randomly selected animals were bled (orbitally). Serum was 
analyzed for IgG specific to DNPOA, as described below. 
At Day 20, all ten animals were anesthetized with an ip injection of 
approximately 0.5 ml of avertin as described above. The animals were 
clipped free of hair on a dorsal area of approximately 2 cm.sup.2 and 
wiped with 70% alcohol. A small incision, approximately 1 cm, was made in 
the clipped area. A hemostat was inserted into the incision and pushed 
anteriorly to the base of the tail and the above-described minipumps were 
inserted. Five animals were implanted with two minipumps each of excipient 
buffer. Five animals were implanted with two minipumps each of IGF-I. The 
rate of delivery for the minipumps gave an IGF-I dose of 120 .mu.g 
IGF-I/day for maximum of 14 days. After recovery from anesthesia, five 
animals each from the excipient and IGF-I groups received a booster ip 
100-.mu.l injection of DNPOA. 
At Day 25, one animal in the excipient group was found dead. 
At Day 34, all nine animals were bled orbitally and the serum was analyzed 
for IgG. 
See Table V for the overall immunization scheme. 
II. Assay of Anti-DNP Antibodies 
IgG: IgG anti-DNP antibodies in the test mouse sera were measured by ELISA 
(enzyme-linked immunoassay) using serum of anti-DNPOA primed mice as a 
reference standard. The ELISA was set up in 96-well plates. Each well was 
coated with 0.1 ml of 2.5 .mu.g/ml DNP.sub.6 HSA (dinitrophenyl human 
serum albumin) for 24 hours at 4.degree. C. 
TABLE V 
______________________________________ 
Retired Male Breeder Mice (BALB/c) Immunized with DNPOA 
1st Injection 
Compound 2nd Injection 
Group Number DNPOA (implant date) 
DNPOA 
______________________________________ 
1 4 Day 0 Excipient 
Day 20 
Day 20 
2 5 Day 0 IGF-I Day 20 
Day 20 
______________________________________ 
After blocking with 0.1% BSA, 0.1 ml of each test sera was added to the 
antigen-coated plates in triplicate and the plates were incubated for two 
hours at room temperature. The plates were washed three times with 
PBS/0.02% Tween 20, and 0.1 ml of 1:2000 dilution of rabbit anti-mouse IgG 
(Cappel Labs) was added to each well. Plates were again incubated two 
hours and washed. Next, 0.1 ml of 1:1600 dilution of goat anti-rabbit 
horseradish peroxidase conjugated antiserum was then added to each well 
for one hour at room temperature. After washing, 0.1 ml of 0.2 mg/ml 
orthophenylene diamine (OPD), 0.01% hydrogen peroxide in 0.05M citrate 
buffer was added to each well, the reaction was stopped with 2M sulfuric 
acid after 30 minutes, and the optical density was read at 490 nm on a 
Microtect plate reader. 
III. Assay of Total IoG 
IgG antibodies in the test mouse sera were measured by an ELISA using 
murine IgG as a reference standard. The ELISA was set up in 96-well 
plates. Each well was coated with 0.1 ml of 1:200 goat anti-murine IgG-Fc 
specific.(Cappel Labs, Westchester, PA) for 24 hours at 4.degree. C. After 
blocking with 0.1% BSA, 0.1 ml of each test sera was added to the 
antibody-coated plates in triplicate and the plates were incubated for 2 
hours at room temperature. The plates were washed three times with 
PBS/0.02% Tween 20, and 0.1 ml of 1:250 dilution of horseradish 
peroxidase-conjugated Fab-specific goat-anti-mouse IgG (Cappel Labs) was 
added to each well. Plates were again incubated two hours and washed. 
After washing, 0.1 ml of 0.2 mg/ml OPD, 0.01% hydrogen peroxide in 0.05M 
citrate buffer was added to each well, the reaction was stopped with 2M 
hydrogen peroxide after 30 minutes, and the optical density was read at 
490 nm on a Microtect plate reader. 
IV. Results 
FIG. 20 shows the concentration of total (FIG. 20B) and OA-specific (FIG. 
20A) IgG in the serum of excipient- or IGF-I-treated mice. IGF-I treatment 
significantly increased the secondary IgG response to antigen at every 
time point examined. While there was a trend toward elevation in total IgG 
levels in the IGF-I group, the values were not statistically increased 
compared to controls. Thus, IGF-I functions to boost the memory response 
of the mammal. It is noted that exposure to IgG after a secondary 
immunization produces a longer improvement in antibody production. 
EXAMPLE III 
Effect on Immune Response After Bone Marrow Transplantation 
The purpose of this experiment is to determine the effects of IGF-I 
treatment of mice on repopulation of the spleen and thymus following bone 
marrow transplantation. 
I. Protocol 
Male BALB/c mice, 19-26 g and 6-7 weeks old (Charles River, San Diego, 
Calif.), were used in the study. The animals were group housed in 
polypropylene cages with food (Purina Rodent Chow 5010, St. Louis, Mo.) 
and water, ad libitum. All animals were weighed the day of pump 
implantation and randomized into groups. Animals were identified by 
stainless-steel ear tags. 
Ten animals per group were studied. Animals were anesthetized with an ip 
injection of approximately 0.4 ml of avertin prior to implantation of 
Alzet osmotic minipumps Model 2002 (0.58.+-.0.03 .mu.l/hr./14 days) filled 
with IGF excipient or 200 .mu.l of rIGF-I described above diluted to 
achieve a daily, continuous delivery of approximately 40 or 120 
.mu.g/day/14 days. 
Daily animal weights were recorded. Twenty-four hours after the implant, 
all animals were irradiated with 900 rads of radiation from .sup.137 
Cesium (4.29 minutes). Within one hour after irradiation animals received 
an intravenous injection of 1.times.10.sup.7 bone marrow cells (250 
.mu.l). 
Femurs and tibias were removed from 40 donor animals. The bone marrow was 
flushed out with PBS. Cells were centrifuged and washed with saline. 
Viable cells were counted and diluted with saline to achieve 10.sup.7 
cell/0.25 ml. 
One half of the animals were sacrificed 14 days after the irradiation 
treatment. All the surviving animals from the group that was irradiated 
and received no bone marrow were sacrificed at this time. The remaining 
animals were sacrificed 23 days after the irradiation treatment. Spleens, 
thymuses, livers, and hearts were removed and weighed. Long bones were 
taken for histology and the spleens and thymuses retained for cytological 
and in vitro assays. Blood was taken for analysis of peripheral cytology. 
The protocol is given in Table VI. 
TABLE VI 
______________________________________ 
Dose of IGF-I 
Group (n) Route (.mu.g/day) 
______________________________________ 
1 10 sc pump 0 no marrow 
2 10 sc pump 0 received marrow 
3 10 sc pump 40 received marrow 
4 10 sc pump 120 received marrow 
______________________________________ 
II. Results 
A. Weight Gain 
Animals not replaced with bone marrow showed a high mortality, where three 
out of ten animals survived for 14 days. For all measures (blood, tissue, 
and whole body) this group of animals showed the expected effect of a 
lethal dose of radiation. 
Animals replaced with bone marrow survived with only two animals out of 30 
dying over the 23-day study. The actual weight gains in the four groups 
are shown in Table VII. 
TABLE VII 
__________________________________________________________________________ 
WEIGHT GAINS 
Thymus Weight (g) 
Spleen Weight (g) 
Day 14 Day 23 Day 14 Day 23 
__________________________________________________________________________ 
No marrow 
8.6 .+-. 0.9 
-- 18.6 .+-. 2.5 
-- 
Marrow only 
12.6 .+-. 1.0 
26.0 .+-. 12.9 
77.8 .+-. 31.5 
74.0 .+-. 29.0 
IGF-I low 
23.5 .+-. 6.2 
36.4 .+-. 9.2 
101.2 .+-. 20.5 
92.0 .+-. 8.3 
IGF-I high 
27.3 .+-. 10.9* 
51.2 .+-. 9.3** 
125.0 .+-. 35.4* 
103.6 .+-. 19.4 
__________________________________________________________________________ 
*p &lt; 0.05 of Marrow Only on same day 
**p &lt; 0.01 
There was a clear effect of IGF-I increasing thymus and spleen weight in 
this model. It appeared that the thymic effect was greater that the 
splenic effect, as there was a maintained doubling of thymus size in the 
high-dose IGF-I group, with the effect on the spleen initially being 
statistically significant, but not maintained at day 23. There was no 
overall effect of treatment on liver or heart weight. 
The dramatic whole body anabolic effect of IGF-I in this setting confirms 
that IGF-I continues to be anabolic on the whole body. The effect of IGF-I 
increasing the mass of the thymus and spleen was surprising in the very 
extreme setting of immune deficiency that this model presents. It might be 
expected in other models of immune deficiency, i.e., AIDS, that IGF-I 
would also show these remarkable efficacies. 
The body weight changes for all four groups are shown in FIG. 21. The 
figure shows clearly the very large weight loss in the animals following 
radiation exposure. There was a clear dose-related effect of IGF-I 
protecting the mice from this catabolism. High-dose IGF-I had a 
significant anabolic effect as early as seven days following treatment and 
this effect persisted to the end of the study. Low-dose IGF-I also caused 
a significant protection at some time points (p&lt;0.05). 
B. Cell Numbers and Mitogenic Responses 
Fourteen days post irradiation, animals receiving 120 .mu.g IGF-I had 
increased numbers of CD.sub.4 +T-cells in the peripheral blood compared to 
control or low-dose IGF-I treatment (FIG. 22). Indeed, the ratio of 
CD.sub.4 to CD.sub.5 increased from 2 to 4 in this treatment group 
compared to controls. These data are consistent with the preferential 
increases in CD.sub.4 cells seen in the spleens of aged mice treated with 
IGF-I for 7 or 14 days. No effect was seen on peripheral B-cell number 
following IGF-I treatment. 
When the splenic lymphocytes from these animals were quantitated by FACS 
analysis, IGF-I treatment was shown to produce a dose-responsive increase 
in the number of T- and B-cells (FIG. 23). However, no effect was seen on 
mitogenic responsiveness of these splenocytes when measured at this time 
point (FIG. 24). 
As was the case with the spleen, the number of lymphocytes repopulating the 
thymus of the IGF-I mice was increased compared to controls (FIG. 28). 
When examined at 21 days post irradiation, IGF-I again induced an 
alteration in the peripheral blood lymphocytes CD.sub.4 :CD.sub.8 ratio 
due to increases in the CD.sub.+ T-cell population (FIG. 25). By this 
time, total splenocyte numbers in the IGF-I-treated groups had returned to 
control values but a slight increase was still measurable in the splenic 
CD.sub.4 +T cell population (FIG. 26). This increase in T-cells was 
reflected in increased mitogenic responsiveness. Con A stimulation of 
splenic T-cells tripled in the high-dose IGF-I-treated mice (FIG. 27). 
B-cell mitogenic responses to LPS were unaffected by IGF-I treatment when 
examined at this time point. 
Surprisingly, the thymic lymphocyte numbers of the high- and low-dose 
IGF-I-treated mice were still dramatically increased compared to controls 
(FIG. 28). 
Taken together with the increases in splenic CD.sub.4 number and Con A 
responsiveness, these data suggest that IGF-I increases the rate of 
peripheral cell repopulation and supports an important therapeutic role 
for this molecule following syngenic bone marrow transplantation. 
EXAMPLE IV 
IGF-I Administration to Man 
This clinical investigation provides evidence that IGF-I also affects the 
immune system of a human. 
I. Protocol 
A Phase I clinical study was conducted of the safety and pharmacokinetics 
following repeat administration (multidose) of IGF-I in healthy adult 
males. Twelve human patients received a bolus injection of 0.03 mg/kg 
rhIGF-I as described above each morning for five consecutive days. On 
screening and ten hours post bolus on day five, blood samples were taken 
for determination of hematology. 
II. Results 
It was found that the hemoglobin, hematocrit, and red blood cells (RBCs) 
were significantly lower on day 5 as compared to screening or 
post-treatment week 2 (p=0.001, 0.0004, 0.0005, and 0.0005). In contrast, 
the white blood cells (WBCs) increased significantly from screening to day 
5 (from 6.1.+-.1.5 to 7.5.+-.1.9 M/CMM, p=0.0018). Furthermore, at 
post-treatment week 2 the WBCs fell significantly from the value at day 5 
(from 7.5.+-.1.9 to 6.4.+-.1.6M/CMM, p=0.003), so that the pretreatment 
and 2-week post-treatment WBC values were not significantly different. 
Therefore, despite the RBCs falling in this study, the WBCs rose. It is 
known that 25 to 30% of the white blood cells are lymphocytes. The 23% 
increase in the total number of WBCs in the blood of the IGF-I-treated 
subjects makes it very likely that there was also an increase in the 
number of lymphocytes following this course of IGF-I treatment in man. 
Compare FIG. 22B, which shows statistically significant changes in the 
peripheral blood CD. +lymphocytes number in mice after treatment with 120 
.mu.g IGF-I. See also Table III on the increased effects of the 
combination of des-IGF-I and bGH on lymphocyte number and WBCs in aged 
rats. 
CONCLUSION 
IGF-I was isolated and named first as a "somatomedin" to indicate that it 
mediated the whole-body growth-promoting activity of GH. It was later 
named IGF-I in recognition of its insulin-like metabolic activities. It is 
therefore surprising that IGF-I, a molecule considered to be a metabolic 
regulator of somatic growth, was shown to have growth factor activity on 
cells of the immune system similar to that of many of the interleukins. 
It is known that GH receptors, IGF-I receptors, and insulin receptors are 
present on cells of the immune system. The functional effect in vivo of 
these receptors and the activity of their ligands on the immune system was 
unknown until the present invention. The effects of insulin and GH on the 
immune system have been taken to be insignificant. See, e.g., Snow, J. 
Immunol., 135:776-778 (1985). Most tissues in the body have receptors for 
GH. IGF-I, and insulin where these hormones act to regulate the basic 
metabolic functions of cells, for example, glucose uptake or amino acid 
transport. The receptors that have been demonstrated to be present in 
immune tissue could function to control these activities, rather than act 
to influence their differentiation, growth, and the immunological 
activities. Recent literature has recognized that the role of IGF-I in 
affecting immune cytology or function is unknown. See Fu et al., J. 
Immunol., 146: 1602-1608 (1991). 
It is well recognized that aged, underfed, or malnourished patients, or 
patients suffering from illnesses or diseases, become immune deficient. It 
is additionally known that these patients also become IGF-I deficient. The 
findings herein suggest that this immune deficiency is directly related 
to, and exacerbated by, if not caused by, this IGF-I deficiency. Restoring 
IGF-I blood concentrations in patients would be expected to result in an 
amelioration of their immune deficiency. IGF-I dramatically affects the 
size of the thymus in several animal models. Thymic growth has been seen 
in hypophysectomized and dwarf rats, in young, adult, and aged rats, in 
mice, and in rabbits. The thymus involutes with age in most animals; it 
reaches a maximal size and then begins involuting in man after puberty. 
This involution is associated with a decline in the activity of the immune 
system. This invention therefore provides in one aspect a means of 
stimulating the immune system of an aged human to restore the thymic 
tissue to that of a much younger person. The combination of an agent that 
has anabolic activity on the major internal organs, with improvement of 
hematology and immune function, makes IGF-I an attractive drug for 
treating adult or aged humans. The ability to rejuvenate the thymus and 
therefore boost the immune system is seen as providing a range of 
therapeutic opportunities. 
Such opportunities include common varied agammaglobulinemia in which 
B-cells fail to mature into Ig secretory cells and the serum contains less 
than 250 mg/dl compared to 1000 mg/dl that is the normal concentration. 
IGF-I produced significant increases in serum Ig levels (FIG. 20) and may 
be useful in this disease. 
A further use of the invention would be to administer the IGF-I to a 
patient who suffers from a hereditary illness that results in an impaired 
immune system. An example of such a patient would be a child suffering 
from congenital thymic aplasia (diGeorge syndrome) in which the thymus is 
atrophied and T-cells are severely diminished, leading to opportunistic 
infections that are often fatal. The reason for this disease is unknown. 
IGF-I might be expected to give an improved size, cellularity, and 
responsiveness of the thymus in these patients. The course of treatment 
would be intermittent, with, for example, a predicted 14-day period of 
treatment being given followed by a resting period of more than 21 days 
between exposures to IGF-I. At this time, the cell counts in the immune 
tissues would have returned to normal, but their ability to response to 
mitogens or to produce antibodies would be enhanced. Such an intermittent 
course of treatment of producing waves of cellular development would be 
sustainable and lead to a long-term restoration of immune function in 
hereditary conditions of the DiGeorge type. 
A third opportunity is acquired immunodeficiency syndrome (AIDS). Patients 
with AIDS have no T-cell immunity and inversed T4/T8 ratios. IGF-I has 
been shown to increase T-cell mitogen responsiveness and specifically 
enhance CD.sub.4 +cell number (FIGS. 5, 10, 11) and as such may be a 
useful drug in the treatment of AIDS. 
The data set forth above suggests that administration of IGF-I is 
beneficial to increase immunoglobulin production in patients suffering 
from insufficient immunoglobulin production. The interval between 
immunizations might be expected to be reduced by the invention herein. The 
more rapid proliferation of cells in vitro from IGF-I-treated mice 
suggested that enhanced antibody responses could be achieved more rapidly. 
This would allow more compressed immunization protocols. For example, in 
man it is common to give primary, secondary, and tertiary immunizations 
separated by many months. During this time the patient is at risk of 
exposure to the agent from which he or she is being protected. It would an 
advantage to reduce the interval between immunizations by using IGF-I to 
boost the immune system so that the above risk could therefore be reduced. 
Another use of the invention is to give a patient a course of IGF-I 
treatment during his or her recovery from major illnesses or following 
surgery when an infection or relapse might be expected. An enhanced immune 
response would be expected to aid such a patient to mount an immune 
challenge to the infection or relapse. 
In the above examples, the effectiveness of IGF-I has been demonstrated as 
follows: (1) in three species (mouse, rat, and rabbit); (2) in both sexes 
(male and female rats); and (3) in several animal models, including 
animals made surgically GH and IGF-I deficient (hypophysectomized rats), 
animals with hereditary GH and IGF-I deficiency (dwarf rats), normal 
animals (ovariectomized rats), normally aged animals that are IGF-I 
deficient (18-month-old rats), animals showing accelerated aging (retired 
breeder mice), and animals with reduced immune function (the aged 
animals). 
It does not necessarily follow from the above studies that a minimum of 14 
days of IGF-I treatment is needed to induce the changes observed. In the 
mouse 14-day treatment was chosen as this proved reliable of inducing 
immune tissue responses. It is possible that 7 days of IGF-I treatment, 
which did induce an increase in cell number, would eventually lead to 
functionally active mature lymphocytes. Additionally, less than 7 days of 
treatment (for example, the 5 days used in Example IV in man) might also 
be an effective period of administration. Furthermore, IGF-I treatment by 
injections rather than continuous infusion is also expected to be 
efficacious. 
It would be reasonably expected that the rabbit, rat, and mice data herein 
may be extrapolated to avians, horses, cows, and other mammals, correcting 
for the body weight of the avian or mammal in accordance with recognized 
veterinary and clinical procedures. Humans are believed to respond in this 
manner as well. IGF-I receptors have been demonstrated on human 
lymphocytes [Kozak et al., Cell Immunol., 109:318 (1987)], and evidence of 
similar responses in man is demonstrated in Example IV. Thus, it would be 
reasonably expected that in man IGF-I would have a beneficial restorative 
effect on immune function in all patients.