PCT WORLD INTELLECTUAL PROPERTY ORGANIZATION [GRAPHIC]
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INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

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(51) International Patent Classification6: (11) International Publication Number WO96/14565
A1
G01N 21/00 (43) International Publication Date: 17 May 1996 (17.05.96)
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(21) International Application Number: PCT/IB95/01019 (81) Designated States: AL, AM, AT, AU, BB, BG, BR, BY,
CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU,
(22) International Filing Date: 18 October 1995 (18.10.95) IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU, LV,
MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO,
(30) Priority Data: RU, SD, SE, SG, SI, SK, TJ, TT, UA, UG, UZ, VN,
08/330,015 27 October 1994 (27.10.94) US European patent (AT, BE, CH, DE, DK, ES, FR, GB,
GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF,
(71) Applicant: OXiGENE, INC. [US/US]; 110 East 59th Street, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD,
New York, NY 10022 (US). TG) ARIPO patent (KE, MW, SD, SZ, UG).

(72) Inventor: PERO, Ronald, W.; Staffan's Grand 6A, S-222 23 Published
Lund (SE). With international search report.
Before the expiration of the time limit for
(74) Agent: DUNHAM, Christopher, C.; Cooper & Dunham amending the claims and to be republished in the
LLP, 1185 Avenue of the Americas, New York City, NY event of the receipt of amendments.
10036 (US).

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(54) Title: METHOD OF TESTING IMMUNE COMPETENCY

(57) Abstract

A method of testing the immune competency of an individual be determining,
from a sample of the blood of the individual, a value for total plasma/serum
thiols including both protein thiols and nonprotein thiols, and comparing the
value so determined with a reference value of total plasma/serum thiols to
ascertain whether the determined value is higher or lower than the reference
value, a determined value lower than the reference value being indicative of
impaired immune function.

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406048.1

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FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of
pamphlets publishing international applications under the PCT.

AT Austria GB United Kingdom MR Mauritania
AU Australia GE Georgia MW Malawi
BB Barbados GN Guinea NE Niger
BE Belgium GR Greece NL Netherlands
BF Burkina Faso HU Hungary NO Norway
BG Bulgaria IE Ireland NZ New Zealand
BJ Benin IT Italy PL Poland
BR Brazil JP Japan PT Portugal
BY Belarus KE Kenya RO Romania
CA Canada KG Kyrgystan RU Russian Federation
CF Central African Republic KP Democratic People's Republic of SD Sudan
CG Congo Korea SE Sweden
CH Switzerland KR Republic of Korea SI Slovenia
CI Cote d'Ivoire KZ Kazakhstan SK Slovakia
CM Cameroon LI Liechtenstein SN Senegal
CN China LK Sri Lanka TD Chad
CS Czechoslovakia LU Luxembourg TG Togo
CZ Czech Republic LV Latvia TJ Tajikistan
DE Germany MC Monaco TT Trinidad and Tobago
DK Denmark MD Republic of Moldova UA Ukraine
ES Spain MG Madagascar US United States of America
FI Finland ML Mali UZ Uzbekistan
FR France MN Mongolia VN Viet Nam
GA Gabon

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406048.1

WO 96/14565 PCT/IB95/01019

METHOD OF TESTING IMMUNE COMPETENCY

BACKGROUND OF THE INVENTION

This invention relates to methods of testing human individuals for
impaired immune function indicative of the presence of, or predisposition to,
diseases associated with compromised immune competency. Such tests may be used,
for example, diagnostically, prognostically, and as a guide in determining the
need for preventive or therapeutic treatment for the disease or condition so
indicated.

More particularly, the invention employs a surrogate measure of DNA repair
activity based on serum/plasma thiol status as a biomarker of human health.
Thus, the method of the invention involves the measurement of chemically
reactive thiols present in naturally occurring amino acids, polypeptides and
proteins found in human serum or plasma. The concentration of these thiols can
predict DNA repair capacity and immune cell responsiveness, and they are
therefore useful indicators of disease progression where impaired immune
function is an essential component of the disease. HIV infection, AIDS, cancer
and autoimmune disorders are examples of diseases that have immunological
components.

European patent No. 0 229 674 as well as several recently published papers
(Pero et al, Carcinogenesis 6:1055-58, 1985; Pero et al, Mutation Res.
142:69-73, 1985; Pero et al, Carcinogenesis 10:693-97, 1989; Pero et al,
Carcinogenesis 10:1657-64, 1989) disclose that DNA repair in general, and
specifically the quantitative estimation of adenosine diphosphate ribosyl
transferase (ADPRT) , is a useful endpoint to estimate health risks in the
detection, prevention and treatment of human chronic age-associated diseases
such as cancer, conditions that predispose to cancer, and autoimmune diseases.
In another aspect, cellular ADPRT activity has been shown to relate to immune
cell responsiveness (Scouvassi et al, Carcinogenesis 8:1295-1300, 1987; Pero et
al, J. Neurosurg. 77:601-06, 1992; Johnstone and Williams, Nature 300:368-79,
1982; Johnson et al, Int. J. Biochem. 22:67-73, 1990), and both these
parameters have been shown to be modulated by the cellular

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reduction/oxidation (redox) balance thought to be in turn mediated by the thiol
containing peptide, glutathione (Pero et al, Cancer Det. Prevent. 14:555-61,
1990; Pero et al, Cancer Res. 50:4619-25, 1990; Fidelius et al, Exp. Cell Res.
170:269-75, 1987; Fidelius and Tsan, Immunology 61:503-08, 1987; Fischman et
al, J. Immunol. 127:2257-62, 1981; Hamilos and Wedner, J. Immunol. 135:2740-47,
1985).

Glutathione exists in the millimolar range within cells (Kosower, Int.
Rev. Cytol. 54:109-60, 1978; Meister, Science 220:472, 1983) and as such it is
believed to be the primary cellular reductant protecting cells from oxidative
cellular injury. However, glutathione levels in human serum/plasma (i.e. 2-27
umoles/liter in Buhl et al, The Lancet, 1294-98, December 2, 1989; Ayers et al,
Anal. Biochem. 154:186-93, 1986) represent only a minor portion of the total
reactive thiol groups present because the proteins in serum/plasma constitute
the major source of reactive thiol groups (i.e. 113-133 umoles/liter in Ellman,
Arch. Biochem. Biophys. 82:70- 77, 1959 and Ayers et al, Anal. Biochem.
154:186-93, 1986). Therefore, the art teaches that there are at least two
distinct classes of thiols in serum/plasma and other biological tissues; namely
protein thiols and nonprotein thiols. A review of the literature supports that
conventional procedures for the analysis of serum/plasma thiols in relation to
human health consequences are based on the analysis of nonprotein thiol sources
such as glutathione or cysteine where protein thiols are excluded from the
analysis by the assay procedure or removed by precipitation using agents such
as trichloroacetic acid, metaphosphoric acid, sulfosalicylic acid or perchloric
acid before any analysis of nonprotein thiols is undertaken (Beutler and
Gelbert, J. Lab. Clin. Med. 105:581-04, 1985; Buhl et al, The Lancet, 1294-98,
December 2, 1989; Eck et al, Biol. Chem. Hoppe Seyler 370:101-81, 1989;
Burgunder et al, Eur. J. Clin. Invest. 18:420-24, 1988; Burgunder and
Lauterburg, Eur. J. Clin. Invest. 17:408-14, 1987; Mimic-Oka et al, Biochem.
Med. Met. Biol. 39:48-54, 1988; Martensson, Metabolism 35:118-21, 1986;
Vendemiale et al, J. Hepatology 9:359-65, 1989). Nonprotein thiol analysis of
biological samples has evolved as the standard assay procedure principally
because of the strong scientific belief that glutathione, a nonprotein thiol,
is the primary cellular reductant protecting cells against the harmful health
effects of oxidant injury (Meister, Science 220:472, 1983).

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Oxidative cellular damage has been postulated to be an important factor in
(i) ageing (Harmon, Age 7:111-31, 1984), (ii) diabetes (Wilson et al,
Diabetologia 27:587-91, 1984), (iii) drug resistance (Spitz et al, J. Cell
Physiol. 156:72-9, 1993), HIV+/AIDS (Baruchel and Wainberg, J. Leuk. Biol.
52:111-14, 1992), (iv) initiation and promotion of cancer (Marnett,
Carcinogenesis 8:1345-73, 1987; Cerutti, Science 227:375-81, 1985), (v)
etiology of cardiovascular and autoimmune diseases (Cross et al, Ann. Int. Med.
107:526-45, 1987) and (vi) modulation of immune function (Carson et al, J. Exp.
Med. 163:746-51, 1986). Most of this evidence comes from evaluating oxidative
stress by comparing glutathione deficient to glutathione proficient cells. For
example, glutathione (or cysteine, its synthetic precursor) deficiency has been
shown to (i) predispose cells to increased sensitivity to DNA damage (Edgren et
al, Int. J. Rad. Biol. 40:355-63, 1985; Valis, The Lancet 337:918-19, 1991),
(ii) inhibit DNA repair (Pero et al, Cancer Res. 50:4619-25, 1990; Edgren and
Revesz, Int. J. Rad. Biol. 48:207-12, 1985), or (iii) induce immune cell
response deficiency (Hamilos and Wedner, J. Immunol. 135:2740-47, 1985;
Fischman et al, J. Immunol. 127:2257-62, 1981; MacDermott et al, Immunology
57:521-26, 1986; Droege et al, Immunobiology 172:151-56, 1986; Stacey and
Craig, Experienta 45:180-81, 1989). In other words, the art teaches that the
nonprotein thiol component is the important factor relating oxidative cellular
damage to human disease development, and the protein thiol component, which
quantitatively dominates in biological samples, has no direct or regulatory
relevance to the health consequences of redox imbalance, and if it indicates
anything at all, it is an indirect and nonspecific estimate compared to the
major regulatory role of the nonprotein thiol component.

Additional evidence for this interpretation is taken from the medical
literature where serum/plasma thiols have been employed to monitor health
disorders. Malignant disease (Beutler and Gelbert, J. Lab. Clin. Med.
105:581-84, 1985), chronic renal insufficiency (Mimic-Oka et al, Biochem. Med.
Met. Biol. 39:48-54, 1988), glucose mediated insulin secretion (Ammon et al,
Diabetologia 32:797-800, 1989), ethanol ingestion (Burgunder et al, Eur. J.
Clin. Invest. 18:420-24, 1988; Vendemiale et al, J. Hepatology 9:359-65, 1989),
fasting (Martensson, Metabolism 35: 118-21, 1986), HIV infection (Buhl et al,
The Lancet, 1294-98, December 2, 1989), AIDS (Eck et al, Biol. Chem. Hoppe
Seyler, 370:101-08, 1989), and cirrhosis (Burgunder

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and Lauterburg, Eur. J. Clin. Invest. 17:408-14, 1987) represent nearly all the
medical conditions where serum/plasma thiols have been used successfully to
monitor health disorders. In all cases, serum/plasma nonprotein thiols such as
glutathione or cysteine were estimated, and great care was taken to eliminate
protein thiols from the assay procedure. These data clearly indicate that it
was not obvious to one skilled in the art to include serum/plasma protein
thiols in the analyses, or that they might be indicators of the health
consequences of oxidative stress, as good as, or even better than, the
nonprotein thiols.

Congestive heart failure (Belch et al, Br. Heart J. 65:245-48, 1991) and
rheumatoid arthritis (Pullar et al, Br. J. Rheumat. 26:202-06, 1987) are the
only exceptions found in the scientific literature where both serum/plasma
protein and nonprotein thiols were included in the final analyses. However, the
logic behind these exceptions did not indicate that total serum/plasma protein
and nonprotein thiols were a better indicator of the health consequences of
oxidative cellular damage than were serum/plasma nonprotein thiols. Contrarily,
it was postulated in these studies that because serum/plasma albumin was an
important factor to these diseases, and because albumin is the major protein
component of serum/plasma and contains numerous thiol functions, it followed
that estimating total serum/plasma protein and nonprotein thiols was an
effective surrogate measure of the oxidation state of albumin. Therefore, the
inclusion in the serum/plasma thiol assay of nonproteins such as glutathione or
cysteine and proteins other than albumin added no significant methodological
advantage even though they were included in the final analyses and contaminated
the estimation of albumin thiols.

SUMMARY OF THE INVENTION

The present invention broadly contemplates the provision of a method for
testing the immune competency of an individual, comprising the steps of
obtaining a sample of blood of an individual to be tested; determining, from
the sample, a value for total plasma/serum thiols, including both protein
thiols and nonprotein thiols, for the individual; and comparing the value so
determined with a reference value of total plasma/serum thiols to ascertain
whether the value so determined is higher or lower than the reference value, a
determined

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value lower than the reference value identifying the individual as having
impaired immune function of significance in detecting, preventing or treating
health disorders.

The invention embraces the unexpected discovery that when the quantitative
analysis of protein thiols is included in the serum/plasma assay procedure,
there exists a highly significant relationship to the function of cellular DNA
repair, estimated as ADPRT activity, in immune proficient mononuclear
leucocytes. Because DNA repair, and specifically ADPRT activity, estimates cell
functions in response to oxidative cellular damage that can predict risk to
immune dysfunction and age associated diseases as has already been documented
in publications discussed above, this discovery establishes that total (i.e.
protein and nonprotein) serum/plasma thiols serve as a quantitative surrogate
assay for the estimation of DNA repair, immune function and health risk in the
detection, prevention and therapy of human diseases. Therefore, total
serum/plasma sulfhydryl analyses have improved sensitivity and biological
relevance over assay procedures estimating only serum/plasma nonprotein thiols
such as glutathione.

The invention contemplates measuring the total level of serum/plasma thiol
groups present in the protein and nonprotein components, and relating the thiol
level to DNA repair, immune function and to the detection, prevention and
treatment of human diseases such as cancer, AIDS, autoimmune and cardiovascular
disorders. Although the invention in its broader aspects is not limited to
specific procedures for total serum/plasma thiol determination, in illustrative
embodiments of the invention total serum/plasma thiols can be conveniently
determined by spectrophotometric or fluorometric procedures involving the
development of chromophores after reaction with thiols using aromatic
disulfides such as DTNB (5,5'-dithiobis-2-nitrobenzoic acid), organic or
inorganic oxidants such as iodosobenzoic acid, diphenyl picrylphenyl hydrazine,
benzfuroxan, 4,4' dimethylaminodiphenyl carbinol, quinones,
trinitrobenzenesulfonic acid, nitroprusside, ferricyanide, cupric copper,
permanganate, iodine, mercurials, nitrous acid, maleimides, halides, platinum
salts, palladium ions, fluorobenzoxadiazole derivatives, or papain-thiol
sensitive p-nitroanilide reaction (Jocelyn, Methods in Enzymology 143:44-67,
1987; Imai, Methods in Enzymology 143:6775, 1987; Ayers et al, Anal. Biochem.
154:186-93, 1986; Singh et al, Anal. Biochem. 213:49-56, 1993). Concentration
of chromophoric agent, pH,

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incubation time of the reaction mixture, and state of denaturation of protein
structure with agents such as sodium dodecyl sulfate, urea, or guanidinium
chloride are well known variables affecting the quantification of thiols, and
as such, they should be optimally controlled for each chromophoric agent and
not serve as a basis to limit the scope of this invention.

In another aspect, this invention proposes to relate ADPRT activity to the
serum/plasma thiol content. This is logically and theoretically accomplished by
taking advantage of the facts that ADPRT is a thiol containing protein, and at
least some of the thiols are located in the zinc binding domain of the enzyme
which in turn controls its participation in DNA repair (Mazen et al, Nucleic
Acids Res. 17:4689-98, 1989).

Therefore, ADPRT activity is dramatically up and down regulated by
cellular reduction/oxidation balance which in turn is regulated and monitored
by thiol status (Pero et al, Cancer Res. 50:4619-25, 1990) Furthermore, it is
found that there is a natural production of ADPRT inhibitors via normal
metabolic cellular processes which can inhibit DNA repair and immune cell
function. These substances were identified as HOC1 and N-chloramines which are
well known oxidants of thiol (Schraufstatter et al, J. Clin. Invest. 85:554-62,
1990). It is also found that most of the total serum/plasma thiols are
chemically reactive with N-chloramines, and as such, this parameter can be used
as a surrogate indicator of the endogenous cellular production of
HOC1/N-chloramines. Because HOC1/N-chloramines produced as byproducts of
cellular metabolism also inhibit DNA repair (Van Rensberg et al. , Free Radic.
Biol. Med. 11:285-91, 1991) and immune function, the serum/plasma thiols
reacting with HOC1/N-chloramines likewise measure the functional health
consequences of oxidatively stressed cells that can occur in human disorders
such as ageing, autoimmunity, cancer, cardiovascular disease, diabetes, drug
resistance and HIV infection.

There are well known distinct classes of ADPRT activity; namely,
constitutive, induced and activated ADPRT activities. DNA damage is a necessary
cofactor that drives the ADPRT enzymatic activity (Satoh and Lindahl, Nature
356:356-58, 1992). The constitutive ADPRT level reflects the intrinsic or
steady state enzymatic activity in response to endogenous cellular levels of
DNA damage induction. However, the ADPRT activity can be activated by
exogenously supplied DNA damaging agents, such as oxidatively stressing cells
by

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exposure to reactive oxygen species produced by phagocytes or chemical agents
(Pero et al, Cancer Det. Prevent. 14:555-61, 1990), to maximum levels of ADPRT
activity. Consequently, induced ADPRT activity (e.g. in response to oxidative
stress) can be calculated by subtracting the constitutive ADPRT activity from
the activated ADPRT activity. This invention also embraces the discovery that
when ADPRT activity is activated by DNA damage and measured as either activated
or induced ADPRT levels, the plasma thiol levels significantly estimate
mononuclear leucocyte ADPRT enzymatic activity when determined in parallel on
the same blood samples as are used to obtain the plasma samples.

Further features and advantages of the invention will be apparent from the
detailed description hereinafter set forth, together with the accompanying
drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph in which PMA induced ADPRT in 1 x 106 HML and cellular
production of HOC1 are plotted against incubation with neutrophils;

FIG. 2 is a graph having two portions in which PMA induced ADPRT activity
in HML is plotted against uM of Chloramine T and of HOC1, respectively;

FIG. 3 is a three-part graph in which absorbance units at 412 nm are
plotted against H202 activated ADPRT for total plasma thiol groups,
N-chloramine insensitive plasma thiol groups, and N-chloramine sensitive plasma
thiol groups;

FIG. 4 is another graph similar to FIG. 3; and

FIG. 5 is a graph illustrating a serum thiol surrogate ADPRT test for HIV
positive and HIV negative patient groups.

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DETAILED DESCRIPTION

The method of the invention for testing the immune competency of a human
individual comprises the steps of obtaining a sample of the blood of the
individual to be tested; subjecting the sample to an assay for determining a
value for total (protein + nonprotein) serum/plasma thiols for the individual;
and comparing the value so determined to a reference value to ascertain whether
the value so determined is higher or lower than the reference value, a
determined value lower than the reference value identifying the individual as
having impaired immune function.

The assay typically includes initially deriving, from the obtained blood
sample, a sample of serum or plasma, and determining a value for total thiols
(i.e., both protein thiols and nonprotein thiols) in the serum or plasma sample
by a spectrophotometric or fluorometric procedure involving the development of
chromophores after reaction with thiols using a suitable chromophoric agent, as
discussed above. Such procedures, in themselves well known in the art, provide
a measurement or reading, e.g. in absorbance units at 412 nm, representing a
determined value of total plasma/serum thiols for the individual.

The determined value is then compared with a reference value for
indicating whether the individual being tested does or does not have impaired
immune function in accordance with whether the determined value of the
individual's total serum/plasma thiols is below or above the reference value.
That is to say, in accordance with the present invention it has now been found
that, for any procedure for assaying total (protein + nonprotein) serum/plasma
thiols of an individual, there exists a reference value such that a determined
value for an individual's total serum/plasma thiols below that reference value
identifies the individual as having impaired immune function.

The establishment of an appropriate reference value to function as an
indicator of impaired immune function will be readily apparent to persons
skilled in the art from the foregoing description. For instance, the reference
value can be established by testing a number of individuals of known immune
competency (impaired and unimpaired) to determine a range of values of total
serum/plasma thiols of such individuals, and identifying the lower limit of the
range (or selecting a point, related thereto, providing a desired confidence
level of

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impaired or unimpaired immune function determination) as the reference value.
Such a reference value is illustrated in Example 5 described below, as the
lower limit of total serum thiols of an HIV (normal immune competency) patient
group. FIG. 5, representing data obtained in Example 5, shows that among HIV+
patients tested, some had determined values of total serum thiols above this
reference value, and others had determined values of total serum thiols below
the reference value. Within the investigatory period, fatalities occurred only
in the latter (below reference value) group of HIV+ patients and not in the
former (above reference value) group of HIV+ patients, substantiating the
correlation between impaired immune function and determined values of total
serum thiols below the reference value.

One illustrative use of the present method is as a guide to deciding
whether to subject a particular patient to treatment for the condition or
consequences of the impaired immune function so ascertained. For instance, in
the case of HIV+ patients as represented by Example 5, those having determined
values of total serum thiols below the reference value (represented by about
0.25 absorbance units at 412 nm) might be subjected immediately to such
treatment while those having total serum thiol values above the reference
value, indicating as-yet uncompromised immune competency, would not yet need
treatment.

In a specific aspect, the method of the invention may be employed as a
surrogate measure of induced ADPRT activity, which is itself an indicator of
the presence of, or a predisposition to, DNA-associated diseases, as described
for example in European patent No. 0 229 674.

The invention will be further described with reference to the Examples set
forth below, in which the following specific procedures were employed:

Blood component preparation. -- Peripheral blood samples (n = 225) from
apparently healthy volunteers, patients with predisposition for cancer and
cancer patients were obtained by venous puncture and collected into heparinized
vacutainers (143 USP units/10 ml tube). The blood samples were first
centrifuged at 100 X G for 10 min and the platelet rich plasma removed with a
Pasteur pipette. Platelet-poor plasmas to be used in these experiments were
prepared by centrifuging the platelet-rich plasmas at 400 X G for 25 min to
pellet the platelets. Next, the original volume of blood samples were restored
by addition of physiologic saline

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and then they were carefully layered on top of a commercially available density
cushion (1.077 gm/ml, Organon Teknika) before spinning at 400 X G for 25 min.
The human mononuclear leucocytes (HML) were isolated from the interphase zone
of the density gradient, washed by centrifugation using RPMI 1640 medium and
the cell density adjusted for in vitro culturing purposes in the conventional
manner. When both HML and neutrophils were needed, the cell fractions were
simultaneously isolated by layering the blood sample on top of neutrophil
isolation medium (Cardinal Associates) , and carrying out all steps in the
density gradient isolation using Krebs-Ringer phosphate buffer with glucose
(KRPG, pH = 7.4) according to the procedure of Nathan (J. Clin. Invest.
80:1550-60, 1987).

Cytotoxicity. -- Regardless of the isolation method used for blood cell
fractionation, HML were always resuspended in 10-20% serum or plasma
supplemented RPMI 1640 medium, pelleted and then resuspended again in either
physiologic saline or KRPG buffer for treatment with either HOC1 or chloramine
T (Sigma). HOC1 concentration was determined from the e235 = 100 M-1cm-1.
Cytotoxicity was monitored by cellular exclusion of trypan blue (0.2% isotonic
solution + 5% serum) after 15 min incubation with the dye at 37 degrees C. The
cytotoxicity of HOC1 and N-chloramines is well known (Schraufstatter et al, J.
Clin. Invest. 85:554-62, 1990). Hence, it was important to determine that any
biochemical effects on ADPRT activity induced by these agents were not related
to acute cytotoxicity. The experimental conditions outlined above, and used to
collect the data reported on herein, were non-acutely cytotoxic.

HOC1 measurement. -- Mixed cultures of HML + neutrophils were assayed for
the production of HOC1 in the extracellular conditioned medium by removal of
the cells by centrifugation following the incubation period, and immediately
trapping the produced HOC1 with taurine (20 mM). Taurine chloramine was then
quantified spectrophotometrically by using the conversion of I- to I2 (E = 2.29
X 104 M-1 cm-1). Details of this procedure have been described by Weiss et al
(J. Clin. Invest. 70:598-607, 1982).

ADPRT assay. -- The procedure was adapted from the permeabilized cell
technique of Berger (Methods Cell Biol. 20:325-340, 1988) with modifications as
previously described (Pero et al, Carcinogenesis 10:1657-64, 1989). Duplicate
samples of 1 x 106 HML in the presence of 0 to 4 x 106 neutrophils were
cultured in 1 ml of

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KRPG buffer for 30 min at 37 degrees C in the presence of PMA (phorbol-12-
myristate-13-acetate, 25 ng/ml). After this co-incubation, the HML + neutrophil
mixtures were harvested by centrifugation, permeabilized, and ADPRT activity
determined by radiometric procedure as described in detail elsewhere (Pero et
al, Carcinogenesis 10:1657-64, 1989). In other experiments, duplicate HML
samples of 1 X 106 per ml KRPG buffer were directly treated with 0-100 uM dose
ranges of HOC1 or chloramine T for 30 min at 37 degrees C which was then
followed immediately by treatment with a standardized dose of PMA (25 ng/ml)
for another 30 min before analysis of ADPRT activity as already referred to
above.

Plasma/serum thiol determination. -- Plasma samples were collected from
the same heparinized blood samples that were used to determine mononuclear
leucocyte poly ADPRT activity. The samples were stored under liquid nitrogen
until subjected to analysis. Each plasma sample was thawed and centrifuged at
2000 X G to sediment any precipitated fibrin. Two ml 20% plasma in water (i.e.
4:1 dilution of plasma with water) was prepared and 30 ul of 5,5'
dithiobis-(2-nitrobenzoic acid) (DTNIB) was added as a 9.5 mg/ml solution
dissolved in 0.1 M K2HPO4, 17.5 mM EDTA, pH 7.5. The mixture was left to react
at room temperature for 1 hr, at which time the absorbance units at 412 nm
(A412) was measured. Chloramine T (Sigma) dissolved in water was then added at
a final concentration of 40 uM and the A412 again read after 30 min. Total
plasma thiols as well as N-chloramine sensitive and insensitive plasma thiols
were calculated by subtraction of reagent blank values from the values of total
DTNB reactive and N-chloramine reactive thiols. In the study involving HIV+ (n
= 15) and HIV- (n = 13) patients initiated in May 1993, the same procedure was
used except serum samples were analyzed instead of plasma samples. The serum
samples were donated by intravenous drug users attending the Aaron Diamond
Research Center for AIDS. The sera were prepared over a period of years and
biologically banked at -80 degrees until used in this study.

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EXAMPLE I

This Example establishes that human phagocytes (i.e. neutrophils) produce
physiologic relevant concentrations of endogenous ADPRT inhibitors. Under
conditions that permit viable cell culturing, a standardized amount of HML
(human mononuclear leucocytes, 1 X 106) was incubated together with increasing
amounts of neutrophils from 0 to 4 X 106 cells per culture. Neutrophils do not
respond to the induction of DNTA damage by an activation of ADP-ribosylation,
and so these cells do not contribute to the estimation of ADP-ribosylation in
this experiment (Ikai et al, Proc. 5 Natl. Acad. Sci. USA 77:3682-85, 1980).
Next these combined cultures were exposed to PMA (phorbol-12-myristate-
13-acetate) to activate ADP-ribosylation in HML, and to induce the production
of reactive oxygen intermediates by neutrophils. The abundant reactive oxygen
intermediates, hydroxyl radical, super oxide anion, and hydrogen peroxide, are
all well known inducers of ADP-ribosylation (Pero et al, Cancer Det. Prevent.
14:555-61, 1990). The data in FIG. 1 show that when HML + neutrophil ratios
reached 1:2 (X 106 cells/ml), which is comparable to the proportion and
concentration in blood, HML ADP-ribosylation began to become severely
inhibited. The respiratory burst induced by PMA exposure of neutrophils was
monitored by HOC1 production. It was concluded that either the presence of
neutrophils or the production of about 80 uM HOC1 or N-chloramine was
sufficient to cause inhibition of HML ADP-ribosylation.

EXAMPLE 2

The effect of various dosage levels of chloramine T and HOC1 on PMA
induced ADPRT activity in HML was investigated utilizing the procedures
described above for such tests, measuring the PMA induced ADPRT activity for
HML subjected to the various dosages, and comparing the values obtained with
measured control values of PMA induced ADPRT activity in HML from the same
source but with zero dosages of chloramine T and HOC1. The results, represented
in FIG. 2, confirm that HOC1 and N-chloramine are potent naturally occurring
ADPRT inhibitors because they can cause greater than 80% inhibition of HML
(human mononuclear leucocyte) ADPRT activity at doses of 80 uM, which are
levels easily attainable in peripheral blood under

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culture conditions that give negligible cytotoxicity. This Example together
with Example 1 clearly shows that ADPRT inhibitors are naturally produced as a
by-product of the respiratory burst of phagocytes which is a normal function of
this cell type designed to kill infectious agents.

EXAMPLE 3

Utilizing the above-described procedures, values were determined for H202
activated ADPRT activity in HML and for total plasma thiols, N-chloramine
insensitive plasma thiols, and N-chloramine sensitive plasma thiols coming from
the same blood samples (n = 225). Results are represented in FIG. 3. This
Example demonstrates that plasma thiols significantly predict the level of
hydrogen peroxide activated ADPRT activity determined in HML (human mononuclear
leucocytes) coming from the same blood samples. Furthermore, this example shows
that the N-chloramine sensitive plasma thiols give a better correlation than
the N-chloramine insensitive plasma thiols to HML ADPRT activity, and that most
of the N-chloramine sensitive plasma thiols are plasma protein thiols and not
just nonprotein plasma thiols. Consequently, the best surrogate predictor of
ADPRT activity was total protein + nonprotein plasma thiols. HOC1 and
N-chloramines are efficient oxidizers of thiols, and could as such in a
surrogate manner indicate ADPRT activity. The logic linking ADPRT activity to
plasma thiols is based on the facts (1) that ADPRT can be dose dependently up-
and down- regulated by reduced and oxidized glutathione, respectively (Pero et
al, Cancer Res. 50:4619-25, 1990) and (2) that ADPRT has thiol amino acid
constituents in the DNA binding domain of the enzyme which in turn control its
participation in DNA repair (Mazen et al, Nucleic Acid Res. 17:4689-98, 1989).

EXAMPLE 4

Again following the above-described procedures, tests were made to compare
values of H2O2 induced ADPRT in HML with values of total plasma thiols,
N-chloramine insensitive plasma thiols, and N-chloramine sensitive plasma
thiols from the same blood samples. Example 4 extends the knowledge disclosed
in Example 3 to show that plasma thiols can also predict hydrogen peroxide HML
(human mononuclear leucocyte) induced

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ADPRT activity (FIG. 4). Examples 3 and 4 also teach that because the activated
and induced levels of ADPRT directly relate to DNA repair in general, then
plasma thiols can also be used to surrogately estimate DNA repair responses in
HML.

EXAMPLE 5

The aforementioned serum samples of HIV+ and HIV- individuals were assayed
by the above-described plasma/serum thiol determination procedure to determine
values of total serum thiols. Results are plotted on the graph of FIG. 5.

Example 5 confirms that estimating N-chloramine sensitive serum thiols has
clinical utility in that reduced levels indicate HML (human mononuclear
leucocyte) ADPRT deficiency that can lead to accumulation of DNA damage and
inhibition of immune function of importance in the progression of HIV+
infection to AIDS and death. The half-solid squares represent the only deaths
that have occurred as of August 1993. The study was conducted in May 1993.

It is to be understood that the invention is not limited to the procedures
and embodiments hereinabove specifically set forth, but may be carried out in
other ways without departure from its spirit.

CLAIMS
What is claimed is:

1. A method for testing the immune competency of an individual, comprising
the steps of

(a) obtaining a sample of blood of an individual to be tested,

(b) determining, from said sample, a value for total plasma/serum thiols,
including both protein thiols and nonprotein thiols, for said
individual, and

(c) comparing the value so determined with a reference value to ascertain
whether said value so determined is higher or lower than said

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reference value, a determined value lower than said reference value
identifying said individual as having impaired immune function of
significance in detecting, preventing or treating health disorders.

2. A method according to claim 1, wherein the determining step comprises
deriving, from the obtained blood sample, a sample of serum or plasma, and
subjecting the serum or plasma sample to an assay for total thiols including
both protein thiols and nonprotein thiols.

3. A method according to claim 2, wherein the assay comprises a
spectrophotometric or fluorometric procedure involving development of
chromophores after reaction with thiols using a chromophoric agent.

4. A method for testing the immune competency of an HIV+ individual,
comprising the steps of

(a) obtaining a sample of blood of an HIV+ individual to be tested,

(b) determining, from said sample, a value for total plasma/serum thiols,
including both protein thiols and nonprotein thiols, for said
individual, and

(c) comparing the value so determined with a reference value established
by determining values for total plasma/serum thiols for HIV-
individuals, to ascertain whether said value so determined is higher
or lower than said reference value, a determined value lower than
said reference value identifying said individual as having impaired
immune function.

5. A method of testing an individual for the presence of or a
predisposition to a disease associated with DNA damage, comprising

(a) obtaining a sample of blood of an individual to be tested, and

(b) subjecting the blood sample to a surrogate test for activated or
induced activity of adenosine diphosphate ribosyl transferase (ADPRT)
by the steps of

(i) determining, from said sample, a value for total plasma/serum
thiols, including both protein thiols and nonprotein thiols, for
said individual, and

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(ii) comparing the value so determined with a reference value of
total plasma/serum thiols corresponding to a reference level of
ADPRT activity, to ascertain whether said value so determined is
higher or lower than said reference value, wherein said presence
of or predisposition to a disease associated with DNA damage is
indicated if said value so determined is lower than said
reference value.

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FIG. 1
[GRAPHIC]

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FIG. 2
[GRAPHIC]

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FIG. 3
[GRAPHIC]

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FIG.4
[GRAPHIC]

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FIG.5
[GRAPHIC]

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-----------------------------------------
INTERNATIONAL SEARCH REPORT International application No.
PCT/IB95/01019
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A. CLASSIFICATION OF SUBJECT MATTER
IPC(6) :G 01 N 21/00,
US CL :436/64, 86, 119, 120, 164; 435/974
According to International Patent Classification (IPC) or to both national classification and IPC
- -----------------------------------------------------------------------------------------------------------------------------------
B. FIELDS SEARCHED
- -----------------------------------------------------------------------------------------------------------------------------------
Minimum documentation searched (classification system followed by classification symbols)

U.S. : 436/64, 86, 119, 120, 164; 435/974
- -----------------------------------------------------------------------------------------------------------------------------------
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

- -----------------------------------------------------------------------------------------------------------------------------------
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
APS, STN
search terms: HIV or AIDS or immun; plasma or serum or blood; protein or thiol or mercapto
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C. DOCUMENTS CONSIDERED TO BE RELEVANT
- -----------------------------------------------------------------------------------------------------------------------------------

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

- -----------------------------------------------------------------------------------------------------------------------------------

Y JOURNAL OF LEUKOCYTE BIOLOGY, Volume 52, issued July 1992, SYLVAIN 1-5
BARUCHEL ET AL, "The role of oxidative stress in disease
progression in individuals infected by the human immunodeficiency
virus", pages 111-114, see page 112.

Y Chemical Abstracts, A.E. FAUVIER,"Biological indicators of oxidative 1-5
stress in humans", abstract no. 236410, Trace Elem. Free Radicals Oxid,
Dis., [Proc. Int. Congr. Trace Elem. Med. Biol.], 4th (1994), pages
57-80.

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/X/ Further documents are listed in the continuation of Box C. / / See patent family annex.

- -----------------------------------------------------------------------------------------------------------------------------------
* Special categories of cited documents: "T" later document published after the international filing
date or priority date and not in conflict with the
"A" document defining the general state of the art which is application but cited to understand the principle or
not considered to be of particular relevance theory underlying the invention

"E" earlier document published on or after the international "X" document of particular relevance; the claimed
filing date invention cannot be considered novel or cannot be
considered to involve an inventive step when the
"L" document which may throw doubts on priority claim(s) document is taken alone
or which is cited to establish the publication date of
another citation or other special reason (as specified) "Y" document of particular relevance; the claimed
invention cannot be considered to involve an
inventive step when the document is combined with one
"O" document referring to an oral disclosure, use, or more other such documents, such combination being
exhibition or other means obvious to a person skilled in the art

"P" document published prior to the international filing "&" document member of the same patent family
date but later than the priority date claimed

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Date of the actual completion of the international search Date of mailing of the international search report
17 MARCH 1996 11 APRIL 1996

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Name and mailing address of the ISA/US Authorized officer
Commissioner of Patents and Trademarks
Box PCT JAN M. LUDLOW
Washington, D.C. 20231

Facsimile No. (703) 305-3230 Telephone No. (703) 308-0651
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Form PCT/ISA/210 (second sheet)(July 1992)*

406048.1

C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT
- -----------------------------------------------------------------------------------------------------------------------------------
Category* Citation of document, with indication, where appropriate, of Relevant to claim No.
the relevant passages
- -----------------------------------------------------------------------------------------------------------------------------------

Y CANCER RESEARCH, Volume 50, issued August 1, 1990, R. W. PERO ET AL, 5
"Oxidative stress induces DNA damage and inhibits the repair of DNA lesions
induced by N-acetoxy-2-acetylaminofluorene in human peripheral mononuclear
leukocytes:, pages 4619-4625, see entire document.

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Form PCT/ISA/210 (second sheet)(July 1992)*

406048.1