Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Binds hormone or other secreted growth regulatory factor,...
Reexamination Certificate
1994-11-28
2002-07-16
Spector, Lorraine (Department: 1647)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Binds hormone or other secreted growth regulatory factor,...
C424S145100, C530S388230, C530S389200
Reexamination Certificate
active
06419927
ABSTRACT:
RELATED PUBLICATIONS
The applicants are authors or coauthors of two articles directed to the subject matter of the instant invention: (1) [applicants only] “Studies of Endotoxin-Induced Decrease in Lipoprotein Lipase Activity”, J. EXP. MED. 154 at 631-639 (September, 1981, published after Sep. 8, 1981), incorporated herein by reference; and (2) [co-authors with Phillip H. Pekala and M. Daniel Lane]: “Lipoprotein Lipase Suppression in 3T3-L1 Cells by an Endotoxin-Induced Mediator from Exudate Cells”, PROC. NAT'L. ACAD. SCI. 79 at 912-916 (February, 1982, published after Feb. 22, 1982), also incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed generally to methods and associated materials for analysis of the effect and operation of invasive stimuli upon animal hosts, and in particular, is concerned with the mechanism and magnitude of the effect that such invasive stimuli may exert upon the activity of anabolic enzymes present in the host.
2. Description of the Prior Art
Several common physiological and biochemical derangement have been seen in various mammalian hosts responding to variety of invasive stimuli such as bacterial, viral and protozoan infections, as well as tumors and endotoxemia. For example, these responses include fever, leukocytosis, hyperlipidemia, reduced food intake and activity, and other modifications in muscle, white blood cell and liver metabolism. Recently, a hypertriglyceridemia in rabbits infected with a protozoan parasite,
Trypanosoma brucei
was reported by C. A. Rouser and A. Cerami, MOL. BIOCHEM. PARASITOL. 1 at 31-38 (1980). The reported hypertriglyceridemia was accompanied by a marked decrease in the activity of the enzyme lipoprotein lipase (LPL) in peripheral tissues.
LPL activity has been observed by others, and it has been noted that this condition has existed when the human bodywas in shock. See E. B. Man, et al, “The Lipids of Serum and Liver in Patients with Hepatic Diseases”, J. CLIN. INVEST. 24 at 623, et seq. (1945); See also John I. Gallin, et al, “Serum Lipids in Infection”, N.ENGL. J. MED. 281 at 1081-1086 (Nov. 13, 1969); D. Farstchi, et al., “Effects of Three Bacterial Infections on Serum Lipids of Rabbits”, J. BACTERIOL. 95 at 1615, et seq. (1968) S. E. Grossberg, et al., “Hyperlipaemia Following Viral Infection in the Chicken Embryo: A New Syndrome”, NATURE (London) 208 at 954, et seq. (1965); Robert L. Hirsch, et al., “Hyperlipidemia, Fatty Liver and Bromsulfophthalein Retention in Rabbits Injected Intravenously with Bacterial Endotoxin”, J. LIPID. RES. 5 at 563-568 (1964); and Osamu Sakaguchi, et al., “Alterations of Lipid Metabolism in Mice Injected with Endotoxins”, MICROBIOL. IMMUNOL. 23 (2) at 71-85 (1979); R. F. Kampschmidt, “The Activity of Partially Purified Leukocytic Endogeneous Meliator in Endotoxin-Resistant C3H/HeJ Mice”, J. LAB. CLIN. MED. 95 at 616, et seq. (1980); and Ralph F. Kampschmidt, “Leukocytic Endogeneous Mediator”, J. RET. SOC. 23 (4) at 287-297 (1978).
While the existence of “mediators” was at least suspected, the effect, if any, that they had on general anabolic activity of energy storage cells was not known. The presentapplicants suspected that these “mediators” exerted a depressive effect upon the activity of certain anabolic enzymes, whose reduced activity was observed in instances where the host entered the condition of shock in response to invasion. Thus, the relationship of the mediator produced by endotoxin-stimulated peritoneal mouse exudate cells, upon endotoxin-sensitive and endotoxin insensitive mice alike, and the development through this investigation, of a reagent for measuring anabolic enzyme activity, was set forth in Ser. No. 299,932, and the further investigation of this system in conjunction with the 3T3 L1 “preadipocyte” model system, and the corresponding development of methods and associated materials for developing antibodies to the “mediators” as well as screening procedures for the identification and development of drugs capable of controlling the activity of these “mediators” was set forth in application Ser. No. 351,290. The work done to date indicates that a need exists for methodology andassociated diagnostic materials, to enable further investigationof the “mediator” phenomenon to proceed, as well as to provide practical diagnostic tools useful in the treatment of the adversesequclae of infection and concomitant shock.
SUMMARY OF THE INVENTION
In accordance with a first aspect of the present invention, a method for preparing a mediator substance for use in assessing the state of anabolic enzymes in mammals, is disclosed, whichfinds particular utility in the instance where the mammals areundergoing invasive stimuli such as, viral agents, bacteria, protozoa, tumors, endotoxemia and others. In its simplest aspect, the method comprises gathering a sample of macrophage cells from amammal and incubating a portion of the macrophage cells with astimulator material associated with an invasive event. Forexample, the stimulator material may be endotoxin, in the instanceof endotoxemia, trypanosomes, in the instance of the above mentioned protozoan parasite
Trypanosoma brucei
, and others.
While the peritoneal exudate cells illustrated in our present and previous applications exemplify sources for themacrophage cells, it is to be understood that such cells may begathered from other than the peritoneal area, and that the present invention contemplates such variation within its scope.
The macrophage cells and the stimulator material are incubated as indicated, and thereafter, the macrophage cells areinduced to produce a mediator substance capable of supressing theactivity of the anabolic enzymes. Preferably, the inducement ofmediator production is accomplished during the incubation periodwhich may, for example, extend up to about 20 hours. The resulting medium may be appropriately treated to recover the mediator substance, and, for example, may be centrifuged, and the supernatant containing the mediator substance drawn off, or the mediator may be precipitated with a 40-60% solution of ammonium sulfate
As mentioned earlier, the mediator substance has a broad range of effects, including inhibitive effects that have beenobserved with respect to anabolic enzymes such as lipoproteinlipase (LPL), acetyl Coenzyme A carboxylase, fatty acid synthetase and the like. Also inhibitive effects have been found with red blood cell formation, as the mediator substance has been found tobe capable of inhibiting the growth and differentiation of erythroid committed cells, by the suppression of a number of growth and differentiation inducers, such as dimethylsulfoxide (DMSO),hexamethylene bisacetamide, butyric acid, hypoxanthine and thelike, as illustrated later on herein in specific examples.
A further embodiment of the present invention comprises a method for detecting various invasive stimuli by their capability of inhibiting the activity of one or more anabolic enzymes. In this method, a plurality of macrophage cell samples, may beprepared and selectively inoculated with a number of known stimulator materials, each characteristic in its effect upon differing anabolic factors. One of the macrophage samples may be inoculated with material from the presumed situs of the infective stimulus, and all samples may thereafter be incubated in accordance with the method described above. Thereafter, testing of each of the supernatants with the mediator substances derived fromthe known stimulator materials, would provide a comparativecontinuum for the identification of any invasive stimulus foundpresent. This testing method may utilize the 3T3 L1 cell system, for example, in the instance where lipoprotein lipase (LPL)activity is utilized as a parameter. Likewise, in the instancewhere red cell inducers are utilized, the Friend virus-transformed erythroleukemia cells may be inoculated and thereafter observed.See Friend, C., Sher, W. Holland J. G. and Sato, G. PROC. NATL.ACAD. SCI. 68, at 378-382; Marks, P. A., Rifkind, R. A.
Cerami Anthony
Kawakami Masanobu
Marshall Gerstein & Borun
Spector Lorraine
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