Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1995-06-07
2001-10-30
Spector, Lorraine (Department: 1647)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C530S388240, C530S387100
Reexamination Certificate
active
06309640
ABSTRACT:
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 body was 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 Intravaneously 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 Mediator 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 present applicants 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 Serial 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 and associated diagnostic materials, to enable further investigation of the “mediator” phenomenon to proceed, as well as to provide practical diagnostic tools useful in the treatment of the adverse sequelae 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 for use in assessing the state of anabolic enzymes in mammals, is disclosed, which finds particular utility in the instance where the mammals are undergoing 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 a mammal and incubating a portion of the macrophage cells with a stimulator material associated with an invasive event. For example, the stimulator material may be endotoxin, in the instance of 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 the macrophage cells, it is to be understood that such cells may be gathered 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 are induced to produce a mediator substance capable of supressing the activity of the anabolic enzymes. Preferably, the inducement of mediator production is accomplished during the incubation period which 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 been observed with respect to anabolic enzymes such as lipoprotein lipase (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 to be 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 the like, 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 be prepared and selectively innoculated with a number of known stimulator materials, each characteristic in its effect upon differing anabolic actors. One of the macrophage samples may be innoculated 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 from the known stimulator materials, would provide a comparative continuum for the identification of any invasive stimulus found present. 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 instance where red cell inducers are utilized, the Friend virus-transformed erythroleukemia cells may be innoculated 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., Terada, M., Ruben, R. C., Gazitt, Y. and Fibach, E. in ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. X. “Hematopoietic Cell Differentiation”. Ed. by D. W. Golde, M. J. Kline, D. Metcalf and C. F. Fox (Academic Press, New York), pp. 25-35 (1978). Naturally, other cellular systems may be utilized in the instance where specific activities may be appropriately observed, and the invention is not limited to the specific cellular systems set forth herein.
The invention includes methods for detecting the presence of samples various invasive stimuli in mammals by measuring mediator substance activity in the mammals.
Cerami Anthony
Kawakami Masanobu
Klauber & Jackson
Spector Lorraine
The Rockefeller University
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