Methods of diagnosis, monitoring and staging of various conditions using IgG antibodies against hydroxy-fatty acid containing sulfatide

A method for screening for, diagnosing, monitoring or staging of multiple sclerosis, HIV infection, rheumatoid arthritis or systemic lupus erythematosus in a subject, is based on the detection of IgG antibodies against the hydroxy-fatty acid form of sulfatide in a tissue or body fluid sample of the subject. The presence of such IgG levels substantially above those in healthy controls is indicative of an increased likelihood that the subject has one of such conditions.

FIELD OF THE INVENTION 
The present invention relates to methods of screening for, diagnosing the 
presence of, or monitoring or staging the course or severity of various 
conditions by detecting the presence of IgG antibodies against the 
hydroxy-fatty acid-containing form of sulfatide in body fluids of such 
individuals, and more particularly to methods of screening for or 
diagnosing the presence of multiple sclerosis (MS), HIV infection, 
rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) in 
subjects suspected of having such conditions, or for monitoring or staging 
the course or severity of MS, RA or SLE by such detection. 
BACKGROUND OF THE INVENTION 
Multiple sclerosis is one of the most common neurological diseases. It 
affects primarily young adults causing visual symptoms, gait disorders, 
limb weakness, sensory disturbances and bowel and bladder dysfunctions. 
Recurrent exacerbations of brain white matter disease disable an 
individual in a stepwise fashion leading to a chronically dependent 
condition. 
Several lines of research point to autoimmune mechanisms as responsible for 
the demyelination that is present in patients with this disease. Treatment 
with steroids and other immunosuppressive compounds often ameliorate the 
signs and symptoms of the disease. Several reports have suggested a role 
for antiglycolipid antibodies in neurodegenerative disorders. See Fredman 
et al., J. Neurol., 238:75-79 (1991); Quattrini et al., J. Neurol. Sci., 
112:152-159 (1992); Pestronk et al., Neurology, 41:357-362 (1991); Ilyas 
et al., J. Neurol Sci., 105:108-11 (1991); and Ryberg, J. Neurol. Sci., 
38:357-382 (1978). It is useful to have as many means as possible for 
diagnosing and/or monitoring conditions such as multiple sclerosis. 
HIV infection is accompanied by neurological disorders affecting both the 
central nervous system (CNS) and the peripheral nervous system (PNS) 
(Simpson et al., Ann Intern. Med., 121:769-785 (1994)). A characteristic 
feature of the nervous system pathology in HIV infection is myelin 
breakdown. See Simpson et al. (1994), supra; Rhodes, Hum. Pathol., 
18:636-643 (1987); Budka, Brain Pathol., 1:163-175 (1991); Shaver, J. 
Neuropathol. Exp. Neurol., 51:3-11 (1992); and Kleihues et al., Acta. 
Neuropathol., 68:333-339 (1985). In HIV leukoencephalo-pathy diffuse white 
matter damage including myelin loss, reactive astrogliosis and appearance 
of scattered multi-nucleated giant cells can be observed (Kleihues et al. 
(1985), supra). To date there is no widely held consensus on the etiology 
of demyelination or other neurological complications associated with HIV 
infection. Proposed mechanisms include direct viral infection of myelin 
and neuronal cells, release of toxic viral proteins and cytokines from 
infected monocytes and macrophages, and the induction of autoantibodies 
(Barnes, Science, 235:1574-1577 (1987); Tillman et al., Seminars in the 
Neurosciences, 3:131-139 (1991); Epstein et al., Ann. Neurol., 33:429-436 
(1993); Solinger et al., Rheum, Dis. Clin. North Am., 17:157-176 (1991)). 
A variety of autoantibodies directed against nucleoantigens, cardiolipin, 
actin, collagen, platelets, erythrocytes, lymphocytes and neutrophils have 
been detected in the sera of HIV.sup.+ individuals (Solinger et al (1991), 
supra). Moreover, antibodies against cerebellar soluble lectin (CSL) and 
myelin basic protein have also been described in the CSL of AIDS patients 
(Hagberg et al., J. Neuroimmunol., 36:245-249 (1992); Saida et al., 
Science, 204:1103-1106 (1979)). 
Studies in rabbits have shown that penetration of antibodies directed 
against the myelin component and glycolipid hapten galactosylceramide (see 
FIG. 1 for structure) across the blood brain barrier results in PNS 
demyelination (Saida et al. (1979), supra). PNS demyelination, in turn, is 
manifested by tremulousness, ataxia, flaccid paresis and limb hypesthesia 
(Saida et al., Ann. Neurol., 9:87-101 (1981)). Otherwise, rabbits with 
high titers of circulating anti-galactosylceramide antibodies did not show 
demyelination, which suggested that the blood brain barrier is protective 
from the antibody damages (Ozawa et al., Acta. Neuropathol., 77:621-628 
(1989)). 
Additional means of diagnosis of HIV infection and the development of PNS 
disease associated with HIV infection are always useful. 
Rheumatoid arthritis and systemic lupus erythematosus are classified as 
autoimmune diseases where the lining of joints (in RA) and the connective 
tissues (in SLE) are affected. These diseases have been found to be 
associated with the aberrant expression of human lymphocyte antigen (HLA) 
in tissues where they normally do not appear, such as in the joint lining. 
As leukocytes penetrate the synovial lining of the joints to cause an 
autoimmune response in RA, affected joints become stiff, sore and swollen. 
Joint immobility and permanent deformity may result from chronic RA. 
Rheumatoid factor (RF) is found in the sera and synovial fluid of adult 
patients with established rheumatoid arthritis. 
In the progression of the disease, SLE is capable of attacking all soft 
internal organs as well as the bones and muscles. This autoimmune disease 
generally manifests itself at the initial stage with a characteristic skin 
rash on the forehead and cheeks. Other common manifestations include hair 
loss, arthritis, severe kidney damage, inflammation of the lining of the 
lungs, accumulation of fluid around the heart, and inflammation of blood 
vessels in the brain. 
Once RA or SLE has begun, the autoimmune diseases are usually associated 
with alternating periods of deterioration and remission. 
Myelin is characterized by a low amount of water, low protein and a high 
lipid content. The solids of myelin are 70-80% lipid and 20-30% protein; 
the lipids of mammalian CNS myelin are composed of 25-28% cholesterol, 
27-30% galactosphingolipid, and 40-45% phospholipid (Basic Neurochemistry, 
Alheus, et al., eds., Little, Brown and Co., Boston, 1st Ed. (1972), page 
376). Table 1 lists the composition of bovine, rat and human myelin 
compared to bovine and human white matter, human gray matter and rat whole 
brain. It can be seen that all the lipids found in whole brain are also 
present in myelin. 
TABLE 1 
__________________________________________________________________________ 
Composition of CNS Myelin and Brain.sup.a 
Gray Whole 
Myelin White Matter 
Matter 
Brain 
Substance.sup.b 
Human 
Bovine 
Rat 
Human 
Bovine 
(Human) 
(Rat) 
__________________________________________________________________________ 
Protein 30.0 
24.7 
29.5 
39.0 
39.5 
55.3 56.9 
Lipid 70.0 
75.3 
70.5 
54.9 
55.0 
32.7 37.0 
Cholesterol 
27.7 
28.1 
27.3 
27.5 
23.6 
22.0 23.0 
Cerebroside 
22.7 
24.0 
23.7 
19.8 
22.5 
5.4 14.6 
Sulfatide 
3.8 3.6 7.1 
5.4 5.0 1.7 4.8 
Total galactolipid 
27.5 
29.3 
31.5 
26.4 
28.6 
7.3 21.3 
Ethanolamine 
15.6 
17.4 
16.7 
14.9 
13.6 
22.7 19.8 
phosphatides 
Lecithin 11.2 
10.9 
11.3 
12.8 
12.9 
26.7 22.0 
Sphingomyelin 
7.9 7.1 3.2 
7.7 6.7 6.9 3.8 
Phosphatidylserine 
4.8 6.5 7.0 
7.9 11.4 
8.7 7.2 
Phosphatidylinositol 
0.6 0.8 1.2 
0.9 0.9 2.7 2.4 
Plasmalogens.sup.c 
12.3 
14.1 
14.1 
11.2 
12.2 
8.8 11.6 
Total phospholipid 
43.1 
43.0 
44.0 
45.9 
46.3 
69.5 57.6 
__________________________________________________________________________ 
.sup.a From W. Norton, in G. J. Siegel et al (eds), Basic Neurochemistry, 
3rd ed., Boston: Little, Brown, 1981, p. 77. 
.sup.b Protein and lipid figures in % dry weight; all others in % total 
lipid weight. 
.sup.c Plasmalogens are primarily ethanolamine phosphatides. 
Sulfatide is galactosylcerebroside esterfied to sulfate at the 3' position 
of the galactose moiety (see FIG. 1 for structure). Many different fatty 
acids are formed in cerebrosides and sulfatides, as well as in other 
sphingolipids. Cerebrosides generally contain very long chain normal 
(lignoceric (24:0) and nervonic (14:1 (.omega.-9))), .alpha.-hydroxy 
(cerebronic (24h:0)), and odd number (23:0, 23h:0) fatty acids (Agranoff 
et al., in Basic Neurochemistry: Molecular, Cellular and Medical Aspects, 
Siegel et al., eds., Raven Press, New York, 5th Edition, 1994, pp. 
97-116). The fatty acid patterns of cerebrosides and sulfatides are the 
same in cerebral cortex as in white matter, varying only slightly with 
maturation of the brain. The percentage of 2-hydroxy acids increases 
slightly, from 50% at birth to 60% at 2 years of age for cerebrosides and 
from 20% to 35% for sulfatides (Svennerholm et al., Brain Research, 
55:413-423 (1973)). 
Antibodies against sulfatide have been found in some patients with a few 
specific autoimmune chronic liver diseases (Toda et al., Hepatology, 
12:664-670 (1990)) and in patients with certain forms of polyneuropathy 
(Pestronk et al., Neurology, 41:357-362 (1991)). Ryberg (1978), supra, 
reported the presence of antisulfatide IgG in one serum and one 
cerebrospinal fluid (CSF) from two different MS patients out of a total of 
60 MS patients tested. The single serum antibody sample found was also 
tested by complement fixation against both forms of sulfatide separated by 
conventional chromatography techniques, and was found to be reactive with 
both the hydroxy fatty acid and non-hydroxy fatty acid forms of sulfatide. 
The highly non-specific results of Ryberg do not suggest that the presence 
of IgG antibodies preferential to the hydroxy fatty acid form of 
sulfatides can serve as a diagnosis of an increased likelihood that the 
subject tested has MS. 
Quattrini (1992), supra, reported increased anti-sulfatide antibody titers 
in one of 17 subjects with MS. The increased titer was selective for IgM 
lambda. No increase in IgG titer was detected. This research was performed 
using an ELISA technology and only used thin layer chromatography (TLC) 
immuno-staining to confirm the positive serum. 
Ilyas reports a study in which human sera is assayed for the presence of 
antisulfatide antibodies to determine if there is a correlation between 
antisulfatide antibodies and various neurological diseases. Sera were 
tested from 53 patients with acute Guillain-Barre syndrome (GBS), 15 
patients with chronic inflammatory demyelinating polyneuropathy (CIDP), 13 
patients with other neurological diseases (OND), 33 patients with 
non-neurological inflammatory, infections, allergic or autoimmune (IIAA) 
diseases (including four with HIV infection), and 31 healthy control 
subjects. The sera were screened for the presence of antibody by ELISA and 
by TLC overlay. The values of anti-sulfatide IgM and IgG were not elevated 
as compared to disease and healthy control sera. However, sera from 11 of 
53 GBS patients had anti-sulfatide IgM levels that were more than 2 SD 
above the mean of the healthy control group as compared to 6 of 92 disease 
and healthy controls. Only 5 of 53 GBS sera had elevated levels of 
anti-sulfatide IgG. Four of 31 IIAA patients showed elevated 
anti-sulfatide IgG antibodies, but there was no report of which IIAA 
patients had such elevated levels, i.e., whether or not the anti-sulfatide 
antibody titers were raised for the HIV patients was not reported. The TLC 
tests conducted for certain sera found positive in the ELISA test did not 
indicate whether or not there was a preference to either fatty acid form 
of sulfatide, although it was noted that even antibodies which reacted 
very strongly with sulfatide in ELISA did not react with sulfatide on TLC 
until more than 10 .mu.g of sulfatide was loaded. 
Fredman conducted TLC-ELISA assays for anti-sulfatide antibodies in the 
sera of 23 patients with GBS, 15 patients with CIDP, and 40 healthy 
controls. Antibodies to sulfatide were observed in 65% and 87% of the sera 
from patients with GBS and CIDP, respectively, but only in 15% of the 
control sera. The antibodies to sulfatide were all IgG. In the control 
both IgM and IgG anti-sulfatide antibodies were detected. No distinction 
between the fatty acid form of sulfatide was drawn other than to recognize 
that in the materials used, the sulfatide contained 20% hydroxy acids. 
Other reports have shown the presence of serum antisulfatide antibodies in 
patients with autoimmune rheumatic disease (Aotsuka et al., Clin. Exptl. 
Immunol., 87:438-443 (1992)), idiopathic thrombocytopenia purpura (Van 
Vleet et al., Brit. J. Haematol., 67:103-108 (1987)), kidney diseases 
(Kikkawa et al., Nippon Jinzo Gakkai Shi, 33:635-642 (1991) and Tamaoki et 
al., Nippon Jinzo Gakkai Ski, 33:1054 (1991)), and diabetes (Buschard et 
al., Lancet, 342:840 (1993)). 
Murakami et al, J. Biol. Chem., 266:15414-15419 (1991), disclose obtaining 
a human monoclonal antibody derived from the lymphocytes of a lupus 
patient with thrombocytopenia. This antibody reacted with single stranded 
DNA and platelets. It was determined that the structure of the platelet 
component that is the antigen target of this monoclonal antibody is a 
monogalactosyl sulfatide. 
SUMMARY OF THE INVENTION 
It has now been discovered that IgG antibodies preferential to the hydroxy 
fatty acid form of sulfatide appear in patients with various conditions, 
specifically including MS patients, HIV infected patients, RA patients and 
SLE patients. A polyclonal antibody which preferentially reacts with the 
hydroxy fatty acid containing form of sulfatide and which is of IgG 
immunotype appears in the serum and other antibody containing fluids and 
tissues of patients having such conditions. 
In order for the assay to be as selective and specific as possible, it is 
important that the assay be conducted under conditions which do not 
degrade the results. For example, assay methods which result in high 
levels of background binding render the results inaccurate and difficult 
to interpret. It is believed that this is one reason why certain prior art 
experiments appear to be substantially less selective for patients with MS 
and HIV infection. It is thought that this is due to the fact that 
conventional ELISA assays are conducted on plastic substrates and either 
the sulfatide antibodies might react with the plastic, or other endogenous 
antibodies present may react with the blocking agent (usually bovine serum 
albumin) giving a false positive result, or both. 
One way to avoid such high levels of background binding is to use the high 
performance thin layer chromatography (HPTLC) immunostaining technique. 
This technique has the further advantage of permitting one to specifically 
identify preferential binding of the antisulfatide IgG reactivity toward 
the hydroxy fatty acid form of sulfatide as opposed to the non-hydroxy 
fatty acid form thereof, even in the presence of antibodies against the 
blocking agent. 
By means of the assay of the present invention, in a large sampling (178) 
of multiple sclerosis patients, 83% (143) showed higher levels of 
antisulfatide IgG specific for the hydroxy fatty acid form of sulfatide, 
as compared to control populations. Furthermore, 33 out of 37 HIV-1.sup.+ 
individuals (89%) tested positive for the presence of serum antisulfatide 
IgG specific for the hydroxy fatty acid form of sulfatide at a 1:1,000 
dilution. In contrast, only 16% of asymptomatic HIV.sup.- controls tested 
positive for the same antibody at the same dilution. High levels of 
specificity for RA and SLE were also found. These results are unexpected 
in light of the prior art experiments discussed above. The preferential 
specificity for the hydroxy fatty acid form of sulfatide could not have 
been predicted and provides another layer of specificity to the assay of 
the present invention. Furthermore, the specific results with respect to 
multiple sclerosis, HIV infection, rheumatoid arthritis and SLE could not 
have been predicted from the experiments which had previously been 
reported in the prior art.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
It has now been discovered that the body fluids of patients having various 
conditions, particularly including multiple sclerosis, HIV-1 infection, 
rheumatoid arthritis, or SEE, contain IgG antibodies specific for the 
hydroxy fatty acid form of sulfatide, while healthy HIV-1 negative 
individuals with no neurological degeneration lack substantial amounts of 
such antibodies. Accordingly, assay methods have been developed for 
diagnosing patients having such conditions which can be routinely 
accomplished using known method steps based on the teachings and guidance 
presented herein without undue experimentation. 
Body fluid or tissue samples, or extracts thereof, of individuals suspected 
of having such conditions, may include sera, CSF, urine, lymphatic fluid, 
saliva and tears. 
According to methods of the present invention, the specified antibodies can 
be detected in a sample of a body fluid of an individual by showing 
preferential reactivity towards the hydroxy-fatty acid-containing form of 
sulfatide or towards anti-idiotypic antibodies thereto, including chimeric 
antibodies, made according to known method steps; see, e.g., Harlow and 
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, N.Y. 
(1988); and Ausubel et al., eds., Current Protocols in Molecular Biology, 
Wiley Interscience, N.Y. (1987; 1992); Sambrook et al. Molecular Cloning: 
A Laboratory Manual., Second Edition, Cold Spring Harbor Press, N.Y. 
(1989) which are incorporated entirely herein by reference. 
It is important that the assay conducted be able to specifically recognize 
antibodies against the hydroxy-fatty acid form of sulfatide. Sulfatide 
obtained from commercial sources such as from Sigma Chemical Company, St. 
Louis, Mo., or that obtained from the urine of patients with conditions 
known to secrete the sulfatide into the urine, such as late infantile 
metachromatic leukodystrophy, may be used as the source of sulfatide as 
long as some means is present to separate the fatty acid forms of the 
sulfatide so that preferential activity against the hydroxy-fatty acid 
form of sulfatide can be observed. HPTLC is an excellent technique for 
this purpose, although it is not the exclusive operable technique. 
More selective results are obtained when assaying specifically for the IgG 
form of antibody. Thus, when immunostaining with either anti-IgG or 
anti-IgM as the second antibody, the results show that the greatest 
activity is with anti-IgG and a weaker reaction with anti-IgM. Thus, while 
the assay technique may be carried out using generic anti-immunoglobulin 
as the second antibody, the strongest and most selective results are 
obtained when using anti-IgG so as to specifically assay for the IgG form 
of antibody against the hydroxy-fatty acid-containing form of sulfatide. 
The anti-sulfatide IgG found in the serum of MS and HIV seropositive, RA, 
and SLE patients reacts equally well with the hydroxy-fatty 
acid-containing fraction of bovine brain sulfatide and that of human 
sulfatide prepared from the urine of a patient with metachromatic 
leukodystrophy. It also recognizes lactosylceramide sulfate (see FIG. 1 
for structure) and to a lesser extent sulfogalactosyldiglyceride 
(seminolipid) (see FIG. 1 for structure). It does not react with the 
nonsulfated parent compound galactosylceramide. No reaction is detected 
with lysosulfatide (see FIG. 1 for structure), indicating that the fatty 
acid is important for binding while changes in the type of lipid moiety, 
as in the seminolipid, greatly reduces but does not abolish the 
reactivity. Reactivity towards lactosylceramide sulfate in which the 
galactose is not directly attached to ceramide is due to the conformation 
of this glycolipid which brings the galactose-3-sulfate and the ceramide 
as close as in the surface molecule. No reactivity is detected with 
sulfoglucuronylparagloboside, a sulfated glycolipid found in PNS myelin. 
The anti-sulfatide moiety was recognized by anti-.kappa. and anti-.lambda. 
light chain antibodies, suggesting its polyclonal nature. 
While a positive indication in the assay of the present invention for IgG 
antibodies against the hydroxy-fatty acid form of sulfatide does not 
guarantee that the subject has a condition such as multiple sclerosis, HIV 
infection, RA, or SLE the presence of such an IgG level substantially 
above that in healthy controls is indicative of an increased likelihood 
that the subject has or may develop MS, AIDS, RA or SLE. Other 
neurological or nephrological conditions may also cause the appearance of 
such antibodies in amounts greater than control. However, in patients 
suspected of having such a condition, the assay of the present invention 
serves as an additional form of diagnosis, as a very large percentage of 
MS patients, HIV positive patients, RA patients and SLE patients have such 
circulating antibodies in amounts significantly greater than in healthy 
controls. Furthermore, a positive result in a cold screening of 
individuals not suspected of having any condition will alert medical 
personnel of the probable existence of a condition which will warrant 
further diagnosis with a substantial likelihood that such condition may be 
MS, RA, or SLE or may indicate the possibility of HIV-1 infection. 
The assay of IgG antibodies which are preferential for the hydroxy fatty 
acid form of sulfatide in patients undergoing treatment or about to 
undergo treatment for MS, HIV infection, RA or SLE may also be used to 
monitor or stage the course of the disease, thereby monitoring the course 
or the efficacy of the treatments. In most cases, the quantitative or 
semi-quantitative results of such an assay, and particularly the trend as 
to whether the titer of such antibodies is increasing or decreasing in a 
given patient, is a factor which is directly related to the efficacy or 
course of the treatment. Thus, as the condition is being ameliorated, the 
antisulfatide antibody titer will generally fall. Similarly, as the 
condition becomes worse, the antisulfatide antibody titer will be expected 
to increase. Accordingly, another embodiment of the present invention is 
the monitoring of treatment or the staging of the condition being treated 
by quantitative or semi-quantitative analysis of the results of assaying 
for IgG antisulfatide antibodies preferential to the hydroxy fatty acid 
form of antisulfatide. 
Another utility for the assay of the present invention is in the testing of 
blood supplies. If a blood sample tests positive for the IgG antibody 
against the hydroxy-fatty acid form of sulfatide, the blood should be 
discarded as probably coming from an unhealthy source and thus possibly 
subject to an unacceptable risk factor for the potential recipient. 
In order to determine whether or not the assay results are substantially 
above those in healthy controls, side-by-side comparisons with the sera 
from known healthy controls may be conducted for a qualitative 
determination. Quantitatively, samples were considered to be positive for 
anti-sulfatide antibodies when titers are equal or greater than either 
1:1,000 or 1:100 for serum and cerebrospinal fluid, respectively. 
While HPTLC on an aluminum backed Silica Gel SG60 HPTLC plate (Merck) is a 
preferred substrate for the assay of the present invention, the method of 
the present invention is not limited thereto. It is believed, however, 
that antisulfatide antibodies may be removed or their reactivity modified 
by certain plastics. Indeed, the sulfatide epitopes may be differentially 
exposed when adsorbed on plastic as compared to silica gel. Thus, the use 
of silica gel as the substrate is preferred in accordance with the present 
invention. 
While the present invention is not limited to the HPTLC technique described 
in the present examples, it is known that some other techniques are 
substantially less effective and so steps should be taken to ensure that 
whatever technique is used, the results are not substantially different 
than that which would be obtained using the HPTLC technique of the present 
examples. Thus, for example, ELISA or other immunoassay techniques may be 
used with sulfatide antigens of defined hydroxy fatty acid composition 
obtained, for example, by chemical synthesis or by previous separation and 
purification from natural sources, possibly including a variation of the 
substrate or the blocking agent to avoid the non-specific binding which 
has been found to occur with certain prior art techniques. Whatever 
immunoassay technique is used, the results should be compared with the 
results using HPTLC to ensure that the technique which is selected does 
not substantially degrade the accuracy of the results. 
Subject to this proviso, any known immunoassay technique can be tested for 
use with the present invention, such as the methods of antibody detection 
described in detail in Harlowe (1988), supra, Ausubel (1987; 1992), supra 
and Sambrook (1989), supra. 
Applicants have not yet confirmed any theory as to why antisulfatide 
antibodies are found in the fluids of patients with such diverse 
conditions. Until there is any further evidence linking the results 
reported herein or confirming any given theory, no such theory would be 
accepted by the scientific community. Hypothetical theories which may or 
may not be applicable are that the antibodies in the sera of such patients 
and identified by the assay of the present invention are generated 
whenever a condition is initiated, co-initiated or exacerbated by an 
immune reaction to a viral invasion. If this were confirmed to be the 
case, the antibodies would be expected to be found in any autoimmune 
condition which may be triggered by an immune reaction, perhaps to an 
invading virus. This theory is supported by initial experimental results 
of the present laboratory (not shown) that the antibody of the present 
invention is also reactive against an antigen on the HIV envelope protein 
and possibly with other HIV viral proteins. 
It should be further understood that while the antibody is denominated as 
"antisulfatide" and specific for the hydroxy fatty acid form of sulfatide, 
this denomination is based on the fact that the assay is conducted against 
chromatographically separated sulfatide molecules. There is no direct 
evidence that the antibodies were actually raised by an epitope on the 
hydroxy fatty acid form of sulfatide. The antibodies may have been 
initially triggered by a different but related sulfoglycolipid and are 
merely cross-reactive with the hydroxy fatty acid form of sulfatide. Thus, 
the use of the terminology antibody "against" a particular antigen, or the 
like, as appearing in the present specification and claims is specifically 
intended to comprehend antibodies which bind to or are reactive with such 
antigen regardless of whether or not that specific antigen was the 
original immunogen which caused the antibody to be raised. 
EXAMPLE 1 
METHOD FOR DETECTING THE PRESENCE OF ANTI-SULFATIDE ANTIBODIES AS AN 
INDICATOR OF HIV INFECTION 
Sulfatide (Sigma, MO) (5 .mu.g/lane) was applied to aluminum-backed Silica 
Gel 60 HPTLC plates (Merck, Darmstadt) and the plates were developed in 
chloroform/methanol/water (65:25:4, v/v/v). The plates were subsequently 
mixed in 0.15% polyisobutylmetachrylate in hexane for 30 seconds, dried, 
and blocked for 1 h in phosphate buffered saline (PBS) containing 2% 
bovine serum albumin (BSA) and 0.05% Tween-20. The plates were incubated 
with a 1:100 dilution of the respective sera samples from human patients 
known to be HIV positive or HIV negative according to a Western blot or 
DNA assay in PBS/BSA for 1 h at room temperature and washed extensively 
with PBS/Tween-20. The plates were then extensively washed as above, and 
incubated with peroxidase-labelled goat anti-human IgG (Boehringer 
Mannheim, Ind.) (1:400 dilution in PBS/BSA) for 1 h. After washing with 
PBS/Tween-20, reactivity towards sulfatide was visualized by incubating 
the plates in 0.01M sodium citrate, pH 6.0, containing 0.2 mg/ml 
4-chloronaphthol and 0.03% H.sub.2 O.sub.2. 
As presented in FIGS. 10A-D, lanes a-c are control HIV-1 negative healthy 
individuals; lanes d and e are HIV-1 positive individuals, without fully 
developed AIDS; lane f is an HIV-1 positive patient with fully developed 
AIDS and demonstrated neurological involvement; and lane g is a sulfatide 
standard, chemically stained with the orcinol/H.sub.2 SO.sub.4 spray 
reagent. The doublet is due to the presence of two sulfatide species 
containing non-hydroxy fatty acids (upper band) and hydroxy fatty acids 
(lower band) respectively. It can be seen that the practice bands in lanes 
d-f all correspond to the hydroxy fatty acid containing species. A method 
of the present invention was thus found to routinely and correctly 
diagnose human patients having no history of neurological or nephrological 
disease as HIV positive or negative. 
EXAMPLE 2 
ANTISULFATIDE ANTIBODY DETERMINATIONS IN THE DIAGNOSIS AND MANAGEMENT OF 
MULTIPLE SCLEROSIS 
Methods: 
Patient Population. Using proper informed consent procedures, a total of 
178 samples were obtained for this study from patients with multiple 
sclerosis at different stages of the disease. This population includes 
patients under .beta.-interferon and steroid treatments. A description of 
the MS population can be seen in Table 2. In addition, serial serum 
samples from 19 MS patients were taken at different time points of 
therapy. As controls, sera from 33 healthy controls and 90 random 
anonymous asymptomatic blood donors were included. Serum samples were also 
analyzed from patients with systemic lupus erythematosus (SLE), hepatitis, 
RF.sup.+ and HIV.sup.+ individuals. The designation of RF.sup.+ denotes 
that the patients were rheumatoid arthritis patients with a positive 
indication of rheumatoid factor in their sera. Description of this latter 
patient population can be seen in Table 3. 
TABLE 2 
______________________________________ 
Serum Antisulfatide IgG in Multiple Sclerosis Patients 
MS Classification 
Abbreviation 
# of Samples 
# Positive 
% Positive 
______________________________________ 
Chronic Progressive 
CP 68 60 88 
Chronic Stable 
CS 36 28 78 
Relapsing RP 19 15 79 
Progressive 
Relapsing Remitting 
RR 22 15 68 
Benign B 4 4 100 
HTLV-1 Positive 
H 2 1 50 
Optic Neuritis 
ON 5 2 40 
Unclassified 
U 22 19 86 
TOTAL 178 144 81 
______________________________________ 
TABLE 3 
______________________________________ 
Serum Antisulfatide IgG* 
Disease Sample # Positive % Positive 
______________________________________ 
HIV.sup.+ 37 33 89 
SLE 11 9 81 
Hepatitis** 
9 5 56 
RF.sup.+ 5 5 100 
MS 207 172 83 
Control*** 
123 20 16 
______________________________________ 
*Analyses performed at 1:1000 dilution by TLCimmunostraining. 
**The hepatitis samples included one sample which was also HIV.sup.+. Thi 
sample was positive for antisulfatide IgG. 
***The control samples included 33 of healthy controls and 90 samples fro 
random asymptomatic blood donors. 
Determination of Antisulfatide IgG. Measurement of antisulfatide IgG was 
performed by HPTLC immuno-staining. Sulfatide was applied onto an 
alumina-backed HPTLC plate and developed in chloroform/methanol/water 
(65:25:4, v:v:v). The plate was then fixed with 0.1% 
polyisobutylmethacrylate in hexane and blocked with 2% bovine serum 
albumin in 50 mM Tris-HCl, pH 7.6, 0.15M NaCl, 0.05% Tween-20 (TBST). The 
plate was then incubated with the test serum previously heat-inactivated 
at 56.degree. C. for 30 min and diluted 1:1,000 in TBST for 1 hr at room 
temperature in polypropylene tubes. After washing with TBST, the plates 
were incubated with peroxidase conjugated anti-human IgG for 1 hr at room 
temperature, and washed as above. The presence of antisulfatide IgG was 
visualized by incubating the plate in 0.2 mg/ml 4-chloronaphthol in 10 mM 
sodium citrate, pH 6.0, 0.15M NaCl, 0.03% hydrogen peroxide. The intensity 
of the staining reaction was visually determined using a scale of 0 to 4 
by two independent observers. 
Results: 
Of 123 control specimens studied, 20 (16%) were positive for the presence 
of antisulfatide antibody. 17 samples were positive from the anonymous 
asymptomatic blood donor population and 3 samples from the known healthy 
control population. Of these 3 latter positive samples, one was from an 
apparently healthy adult male who had Campylobacter infection one month 
prior to testing of his serum. A follow up serum specimen obtained one 
year later was negative. The second specimen was that of an adult male 
with a history of autonomic disturbance of bladder dysfunction and the 
third was from a hospitalized adult female with aseptic meningitis. 
Serum antisulfatide IgG was present in 143/178 samples from multiple 
sclerosis patients. The results are summarized in tables 2 and 3. 
Comparison of the antisulfatide IgG levels between the control and MS 
populations indicated a statistically significant difference (p&lt;0.05) 
(FIG. 2). In general, the chronic form of the disease gave a consistently 
higher reaction than did the other diagnostic categories. In patients with 
optic neuritis (ON), which in some patients is the first sign of the 
disease, 2/5 patients were positive for the presence of antisulfatide IgG 
under the conditions tested. One of the three negative specimens was from 
a patient who had recovered from a single attack of optic neuritis one 
year earlier. It is not known at this point which if any of the five ON 
patients tested went on to develop MS. With steroid treatment in some MS 
patients, there appeared to be a decrease in the levels of antisulfatide 
IgG associated with this form of therapy. See FIGS. 3-6 and 8. However, in 
some other patients, it was not clear that this was the case (see FIG. 9). 
It is believed, however, that longer term study will confirm the 
quantitative correlation of antisulfatide antibody titer with efficacy of 
treatment. FIG. 7 shows the increase in antisulfatide level over time in 
an MS patient suffering from worsening clinical condition. 
Antisulfatide IgG was also found in HIV.sup.+ (89%) as well as in SLE (81%) 
patients and RF.sup.+ (100%) individuals. These data are given in Table 3. 
This table also shows the antisulfatide IgG in 56% of the hepatitis 
patients studied. 
Conclusions: 
1. Higher levels of antisulfatide IgG preferential for the hydroxy fatty 
acid forms of sulfatide are present in 83% of patients diagnosed with 
multiple sclerosis as compared to control populations. Since the MS 
population sample included individuals who were under treatment with 
either .beta.-interferon or steroids, the percent of positivity in MS 
patients is suspected to be initially higher. 
2. Higher levels of antisulfatide IgG preferential for the hydroxy fatty 
acid forms of sulfatide are present in 100% of patients diagnosed with 
rheumatoid arthritis and 81% of patients diagnosed with SLE as compared to 
control populations. 
3. In some patients a decrease in the antisulfatide IgG levels seems to be 
associated with the steroid treatment and in the improvement of the 
clinical condition of the patient. However, in some patients this response 
to steroids was not observed in the serum samples analyzed. 
In summary, a high degree of positivity for antisulfatide IgG in the sera 
of patients with multiple sclerosis, rheumatoid arthritis and SLE has been 
demonstrated, as well as in HIV.sup.+ individuals. In contrast, the 
antibody is detected with much less frequency in the sera of control 
individuals. 
EXAMPLE 3 
INTRATHECAL SYNTHESIS OF ANTISULFATIDE IgG IS ASSOCIATED WITH PERIPHERAL 
NERVE DISEASE IN THE ACQUIRED IMMUNODEFICIENCY SYNDROME 
Materials and Methods: 
Serum and CSF collection. Sera were obtained from 37 HIV seropositive 
(HIV.sup.+) individuals. These included asymptomatic individuals, 
individuals with mild cognitive and/or motor dysfunction and individuals 
with AIDS and CNS degeneration. Cerebrospinal fluid obtained in the course 
of diagnostic evaluations was derived from 11 of the above individuals and 
from 17 additional HIV.sup.+ individuals. Three of these CSF samples were 
from HIV.sup.+ individuals with neurosyphilis. The 123 HIV seronegative 
(HIV.sup.-) control samples included sera form 33 known healthy 
individuals and 90 randomly chosen samples from asymptomatic blood donors. 
The CSF samples used as controls were from 18 HIV.sup.- individuals 
undergoing testing for other unrelated neurological conditions. The 
specimens were routinely heat inactivated at 56.degree. C. for 30 minutes 
prior to use. 
Glycolipids. Bovine brain sulfatide and galactosylceramide were obtained 
from Sigma Chemical Company, St. Louis, Mo. A mixture of sulfatide and 
lactosylceramide sulfate was also obtained from the urine of a two year 
old boy with late-infantile metachromatic leukodystrophy. 
Sulfogalactosyldiglyceride (seminolipid) was obtained from HSC, Toronto, 
Canada. Lysosulfatide was prepared from sulfatide by removal of the fatty 
acid moiety with alkaline base hydrolysis (Dubois et al., Anal. Biochem., 
102:313-317 (1980)). Sulfoglucuronyl-paragloboside was kindly donated by 
Dr Firoze Jungalwala, Shriver Center, Waltham, Mass. 
TLC immunostaining. Sulfatide (5 .mu.g/lane) was applied on aluminum backed 
Silica Gel 60 HPTLC plates (Merck) and the plate developed in 
chloroform/methanol/water 65:25:4 (v:v:v). The plate was cut into 
individual strips and fixed 30 seconds in 1.5% polyisobutylmethacrylate in 
hexane. The strips were than blocked 30 minutes in 50 mM Tris HCI, pH 7.6, 
0.15M NaCl, 0.05% Tween-20 (TBS/Tween) containing 2% BSA. Each strip was 
then incubated with the test serum diluted in TBS/Tween for 1 hr at room 
temperature. The strips were washed with TBS/Tween and incubated 1 hr with 
peroxidase-labeled goat anti-human IgG (Jackson Laboratories, Pa.) and 
washed as above. The strips were then incubated with 0.2 mg/ml 
4-chloronaphthol in 10 mM Na citrate, pH 6.0, 0.03% hydrogen peroxide. For 
the antigenic specificity studies, the glycolipids indicated in FIG. 1 
were applied in equimolar amounts on the HPTLC plate which was then 
developed in chloroform/methanol/water 60:32:7 (v:v:v). The determination 
of the IgG isotype was performed as above except that 
peroxidase-conjugated monoclonal anti-human IgG1, IgG2, IgG3, and IgG4 
(The Binding Site, Birmingham, UK) were used as second antibodies. 
Peroxidase conjugated goat anti-human kappa and lambda light chains used 
to determine the nature of the light chain of the antisulfatide IgG were 
obtained from Fisher Scientific. Chemical staining of the glycolipids was 
performed with the orcinol-H.sub.2 SO.sub.4 spray reagent. 
Purification of antisulfatide IgG. The antibody was purified from 190 ml of 
heat inactivated serum from a healthy HIV.sup.+ individual. The IgG 
fraction was precipitated with 33% ammonium sulfate and purified by 
ion-exchange chromatography. The sulfatide coated-octyl Sepharose affinity 
column was prepared as described in Hirabayashi et al., J. Biochem., 
94:327-330 (1983). An aliquot of the IgG fraction (about 80 mg protein) 
was applied onto the column which was then extensively washed with PBS. 
The bound IgG fraction was eluted with 3M Na thiocyanate in PBS and 
immediately dialyzed against PBS. 
Immunocytochemical analysis of human brain tissue. Immunocytochemical 
staining with the affinity purified antisulfatide IgG was performed on 
frozen human brain section. The tissue sections were incubated for 1 h in 
10% normal goat serum (NGS) in PBS followed by an overnight incubation 
with the affinity purified antisulfatide-IgG diluted 1:500 in PBS 
containing 2% NGS. After three washes in PBS, the sections were incubated 
for 5 hours with peroxidase conjugated anti-human IgG (1:300 dilution in 
PBS/2% NGS). After washing as above, the sections were incubated with 1 
mg/ml 3,3'-diaminobenzidine and 0.005% hydrogen peroxide in 0.05M 
Tris-HCl, pH 7.6 containing 10% (v/v) imidazole. To remove lipids prior to 
immunochemical staining the sections were pretreated with 
chloroform/methanol 2:1 as described in Wikstrand et al., J, Neuropathol. 
Exp. Neurol., 50:756-769 (1991). 
Results and Discussion: 
This experiment was designed to assess the presence in serum and CSF of 
antibodies directed against sulfatide, a major glycolipid component of 
myelin whose structure is closely related to that of galactosylceramide 
(see FIG. 1 for structures). A semi-quantitative high performance thin 
layer chromatography (HPTLC)-immunostaining method was favored to detect 
antisulfatide antibodies in biological fluids over other more quantitative 
procedures, such as enzyme-linked immunoadsorbent assays (ELISA), because 
the HPTLC-immunostaining method yielded no false positive results caused 
by the cross-reactivity of some sera with the blocking agent. Under the 
present assay conditions, samples were considered to be positive for 
antisulfatide antibodies when titers were equal to or greater than either 
1:1,000 or 1:100 for serum and cerebrospinal fluid, respectively. 
Serum samples were first analyzed from HIV-seropositive individuals and 
from HIV-seronegative controls with no apparent symptomatology or with 
non-AIDS related pathologies. Thirty three out of 37 HIV seropositive 
individuals (89%) tested positive at 1:1,000 dilution for the presence of 
serum antisulfatide IgG. In contrast, 20 of 123 sera (16%) from 
asymptomatic HIV seronegative controls tested positive for the antibody. 
The association between HIV seropositivity and presence of serum 
antisulfatide antibodies was statistically significant (two-tailed p&lt;0.01, 
Chi square with Yates correction and Fisher's exact test). Nonetheless, 
among HIV-seropositive individuals, serum antisulfatide IgG levels showed 
no correlations with clinical status parameters (CDC staging or CD4 T cell 
counts). 
In addition, 8 out of 25 HIV-seropositive individuals with neurological 
complications tested positive for CSF antisulfatide IgG (titer &gt;1:100). 
None of 18 age-matched HIV-seronegative controls (including 5 
asymptomatic, 5 with multiple sclerosis, one with SLE, 2 with stroke, 2 
with laryngeal carcinoma, 2 with diabetic polyneuropathy, and one with 
subarachnoid hemorrhage) tested positive for CSF antisulfatide IgG. The 
association between CSF antisulfatide antibody positivity and HIV 
infection with neurological complications was statistically significant 
(two-tailed p&lt;0.01, Chi square with Yates correction and Fisher's exact 
test) when compared to HIV-seronegative individuals. Although HIV-infected 
individuals with no neurological complications would have been a more 
preferable reference group, CSF samples were not available for this type 
of individuals because of lack of clinical indication to obtain them. The 
25 HIV-infected individuals with neurological complications were then 
stratified according to the presence of either predominantly central (18 
individuals) or peripheral (7 individuals) nervous system disease. 
Analysis of the HIV-infected individuals stratified in this way, showed a 
significant association between presence of CSF antisulfatide antibody and 
peripheral nervous system involvement (6/7 with peripheral as compared to 
2/18 HIV-infected individuals with predominantly central nervous system 
disease, p=0.03, Chi square test with Yates correction and p=0.015, 
Fisher's exact test). Two out of 3 additional HIV-infected individuals 
with neurosyphilis showed CSF antisulfatide positivity. 
The statistical significance of the association between HIV infection with 
neurological complications and CSF antisulfatide antibody positivity, as 
well as the presence of CSF antisulfatide antibody positivity in AIDS 
patients with neurosyphilis, prompted the determination of whether 
antisulfatide antibodies were synthesized intrathecally or were derived 
from peripheral blood through blood-brain barrier leakage. Paired serum 
and cerebrospinal fluid samples from 11 HIV-seropositive individuals were 
analyzed. The CSF/serum ratio of total IgG were used to standardize 
CSF/serum antisulfatide antibody ratios (Table 4). A standardized 
CSF/serumratio of up to 1 could be accounted for by peripheral blood 
leakage into the CSF compartment, while a standardized ratio greater than 
1 is indicative of intrathecal synthesis of antibodies. Out of the 11 
samples analyzed, 4 CSF samples had antisulfatide antibody positivity. Out 
of the latter 4, three with normalized CSF/serum antisulfatide antibody 
ratios greater than 10 corresponded to individuals with predominantly 
peripheral nervous system disease (one with ascending radiculopathy, a 
second one with peripheral neuropathy and dementia, and the third one with 
sensory loss below knee in stocking distribution in the left leg). The 
fourth individual with CSF antisulfatide antibody positivity but 
normalized CSF/serum ratio of less than 1, corresponded to an AIDS 
individual with neurosyphilis. Higher levels of antibody in the CSF were 
observed and appeared to correlate with peripheral neuropathy. 
TABLE 4 
__________________________________________________________________________ 
Antisulfatide IgG Levels and Standardized Antisulfatide IgG Ratios in 
AIDS Patients with 
Neurological Involvement 
AS IgG 
Predominant 
AS.sup.a IgG 
in Serum 
Standardized 
neurological 
in CSF 
CSF IgG 
Serum 
IgG AS Ig Ratios 
# Diagnosis 
disease 
Titer 
mg/dL 
Titer 
mg/dL 
(CSF/Serum).sup.b 
__________________________________________________________________________ 
1 ascending 
Peripheral 
1:100 
2.8 1:1,000 
730 
26 
radiculopathy 
2 peripheral 
Peripheral 
1:1,000 
89.1 1:1,000 
1163 
13 
neuropathy, 
dementia 
3 sensory neuropathy 
Peripheral 
1:100 
2.3 1:10,000 
2397 
10 
left leg 
4 neurosyphilis 
Peripheral 
1:100 
137.0 
1:10,000 
3815 
0.3 
5 dementia Central 
&lt;1:100 
7.8 1:1,000 
942 
0 
6 seizures Central 
&lt;1:100 
10.3 1:100 
2024 
0 
7 seizures, liver 
Central 
&lt;1:100 
10.9 1:1,000 
2949 
0 
carcinoma 
8 minor brain 
Central 
&lt;1:100 
8.4 1:100 
ND.sup.c 
0 
atrophy 
9 headaches 
Central 
&lt;1:100 
6.8 1:10,000 
1667 
0 
10 
B-cell lymphoma, 
Central 
&lt;1:100 
7.1 1:10,000 
2122 
0 
multiple brain 
lesions 
11 
seizures, 
Central 
&lt;1:100 
4.3 1:100 
1215 
0 
dementia 
__________________________________________________________________________ 
.sup.a Antisulfatide; 
.sup.b Standardized AS IgG ratios = (AS IgG in CSF/AS IgG in serum)/(Tota 
IgG in CSF/Total IgG in serum); 
.sup.c Not done 
Based on the results presented above, CSF antisulfatide antibody positivity 
in CSF/serum ratios greater than 10 is therefore significantly associated 
with peripheral nervous system disease in HIV-infected individuals 
(two-tailed p.ltoreq.0.01, Chi square with Yates correction and Fisher's 
exact test). The remaining 7 paired samples, with serum but not CSF 
antisulfatide antibody positivity, corresponded to HIV-infected 
individuals with predominantly central nervous system involvement. These 
results illustrate the protective role of the blood-brain barrier to the 
peripheral nervous system-oriented effects of antisulfatide antibodies in 
the CSF compartment. However, an event causing disruption in the 
blood-brain barrier would allow leakage of antisulfatide antibody into the 
CSF compartment from the peripheral blood as was observed in the AIDS 
patient with neurosyphilis. 
To further characterize the antisulfatide IgG, antibody was purified by 
affinity chromatography through octyl-Sepharose coated with sulfatide 
(Hirabayashi (1983), supra). Serum, but not CSF, from an HIV-infected 
individual was used for the antibody affinity purification due to sample 
size limitations. The antigenic specificity was examined by HPTLC 
immunostaining using the glycolipids indicated in FIG. 1. Both the 
original serum and the affinity purified IgG were analyzed and showed the 
same specificity. The results of this analysis are shown in FIG. 3, panels 
C (serum) and D (affinity purified IgG). The antisulfatide IgG reacted 
equally well with bovine brain sulfatide (lane b) and human sulfatide 
prepared from the urine of a patient with metachromatic leukodystrophy 
(lane e, upper band). It also recognized lactosylceramide sulfate (lane e, 
lower band) and to a lesser extent, sulfogalactosyldiglyceride 
(seminolipid) (lane d). It did not react with the non-sulfated parent 
compound galactosylceramide (lane a), suggesting that galactose 3-sulfate 
is part of the epitope recognized by the antibody. As mentioned above, the 
antibody reacted preferentially with the hydroxy-fatty acid containing 
form of sulfatide. No reaction was detected with lysosulfatide (lane c) 
indicating that the fatty acid is important for binding, while changes in 
the type of lipid moiety, as in the seminolipid, greatly reduced but did 
not abolish the reactivity. Reactivity towards lactosylceramide sulfate, 
in which the galactose is not directly attached to ceramide, is due to the 
conformation of this glycolipid which brings the galactose-3-sulfate and 
the ceramide as close as in the sulfatide molecule (Fredman et al., 
Biochem. J., 251:17-22 (1989)). No reactivity could be detected with 
sulfoglucuronylpara-globoside, a sulfated glycolipid found in PNS myelin 
(Chou et al., J. Biol. Chem., 261:11717-11725 (1986), Ilyas et al., J. 
Neuroimmunol., 37:85-92 (1992)) (results not shown). The antisulfatide 
antibody was recognized by anti-.kappa. and anti-.lambda. light chain 
antibodies suggesting its polyclonal nature. Isotype determination 
indicated that the antisulfatide antibody was predominantly of the IgG1 
type. 
To establish whether the antisulfatide IgG could recognize and bind to its 
antigen in situ, frozen human brain sections were immunostained with the 
affinity purified antibody. The antibody was found to stain preferentially 
oligodendrocytes and their processes associated with axons in the human 
pontine white matter. Pre-treatment of the sections with 
chloroform/methanol (2:1) to remove glycolipids, greatly reduced the 
staining, suggesting that the main target of the antibody in the section 
was indeed a glycolipid in nature. The preferential staining of the 
oligodendrocytes was consistent with the high levels of sulfatide in these 
cells (Norton et al., J. Neurochem., 21:759-773 (1973)). 
In terms of the mechanisms for antisulfatide antibody-mediated 
pathogenesis, it is worth mentioning that sulfatide, besides being a major 
component of myelin (Norton et al. (1973), supra), is also present in the 
kidney and at lower concentrations in spleen, erythrocytes, granulocytes, 
platelets, endothelial cells, stomach, intestine and testis (Ginsburg et 
al., Biochimie, 70:1651-1659 (1988)). Sulfatides have been suggested to 
play a role in the Na.sup.+ transport (Zalc et al., FEBS Lett., 92:92-96 
(1978)), in the binding of opiates to their receptor (Graves et al., 
Science, 207:75-76 (1980)), and in cell adhesion. Several proteins 
including properdin (Holt et al., J. Biol. Chem., 265:2852-2855 (1990)), 
factor Xl l, the adhesive glycoproteins laminin, thrombospondin and von 
Willebrand factor (Ginsburg et al. (1988), supra) and the members of the 
selectin family L-selectin/LECAM (Suzuki et al., Biochem. Biophys. Res. 
Comm., 190:426-434 (1993)) and CD62/P-selectin (Aruffo et al., Cell, 
67:35-44 (1991)) have been found to interact with sulfatide. The fact that 
sulfatide is a major component of myelin and is also involved in cell 
adhesion suggests that the antisulfatide antibody in the cerebrospinal 
fluid of AIDS patients has the potential to induce myelin damage and to 
inhibit cell function by interference with the binding of sulfatide to its 
ligands. 
The antisulfatide antibodies detected in the CSF of AIDS patients with PNS 
disease are probably synthesized intrathecally by infiltrating peripheral 
blood cells. On the other hand, the presence of CSF antisulfatide in 
patients with neurosyphilis mimics the findings in rabbits where the 
blood-nerve barrier is defective at the dorsal root ganglia and the nerve 
root (Ozawa et al., Acta. Neuropathol., 77:621-628 (1989)). Further 
research should elucidate the histopathogenesis of PNS demyelination 
associated with HIV infection and the presence of antisulfatide 
antibodies. 
All references cited herein, including journal articles or abstracts, 
published or corresponding U.S. or foreign patent applications, issued 
U.S. or foreign patents, or any other references, are entirely 
incorporated by reference herein, including all data, tables, figures, and 
text presented in the cited references. Additionally, the contents of the 
references cited within the references cited herein are also entirely 
incorporated by reference. 
Reference to known method steps, conventional methods steps, known methods 
or conventional methods is not in any way an admission that any aspect, 
description or embodiment of the present invention is disclosed, taught or 
suggested in the relevant art. 
The foregoing description of the specific embodiments will so fully reveal 
the general nature of the invention that others can, by applying knowledge 
within the skill of the art (including the contents of the references 
cited herein), readily modify and/or adapt for various applications such 
specific embodiments, without undue experimentation, without departing 
from the generic concept of the present invention. Therefore, such 
adaptations and modifications are intended to be comprehended within the 
meaning and range of equivalents of the disclosed embodiments, based on 
the teaching and guidance presented herein. It is to be understood that 
the phraseology or terminology herein is for the purpose of description 
and not of limitation, such that the terminology or phraseology of the 
present specification is to be interpreted by the skilled artisan in light 
of the teachings and guidance presented herein.