Patent us20020119453 – ho-1 as a diagnostic and prognostic test for dementing diseases – google patents brain anoxia

Alzheimer’s disease is a neurodegenerative disease which causes dementia. The period from first detection of AD to termination can range from a few years to 15 years, during which time the patient progressively suffers loss of both mental function and control of bodily functions. There is significant variability in the progress of the disease. While the majority of patients have a gradual, inexorable progression (losing on average 3 to 4 points on the 30 point folstein mini-mental state score annually), approximately 30% of AD cases have a prolonged stable initial plateau phase lasting several years (haxby et al., 1992).


A subgroup of patients has a fulminant, rapidly progressive downhill course over several years (mann et al., 1992).Brain anoxia other patients (about 10% of cohorts) remain slowly progressive, showing only gradual decline from year to year (grossi et al., 1988). The pathological, chemical, and molecular bases of this heterogeneity remain undetermined. Recognition of the variability of AD progression represents an important clinical insight, and may explain the diagnostic difficulties presented by “atypical” cases.

Using immunostaining techniques in conjunction with laser scanning confocal microscopy, intense HO-1 immunoreactivity in neurons and astrocytes of post-mortem hippocampus and temporal cortex derived from AD subjects has been observed, whereas neural HO-1 staining was faint or non-existent in the hippocampus and temporal cortex of control specimens matched for age and post-mortem interval (schipper et al., 1995).Brain anoxia furthermore, consistent co-localization of HO-1 to neurofibrillary tangles and senile plaques in the AD specimens has been demonstrated. Finally, robust 32 kda bands corresponding to HO-1 were observed by western blotting of protein extracts derived from AD temporal cortex and hippocampus after SDS-PAGE, whereas control HO-1 bands were faint or absent. These results indicate that HO-1 is significantly over-expressed in neurons and astrocytes of AD hippocampus and cerebral cortex relative to control brains and support the contention that AD-affected tissues are experiencing chronic oxidative stress.

AACD is a term used to identify individuals who experience a cognitive decline that falls short of dementia.Brain anoxia satisfaction of criteria (world health organization) for this diagnosis requires a report by the individual or family of a decline in cognitive function, which is gradual, and present at least 6 months. There may be difficulties across any cognitive domains (although memory is impaired in the vast majority of cases), and these must be supported by abnormal performance on quantitative cognitive assessments for which age and education norms are available for relatively healthy individuals (ie., the patient is compared to normal subjects his/her own age). Performance must be at least 1 SD below the mean value for the appropriate population on such tests. Neither dementia, nor significant depression or drug effects may be present.Brain anoxia no cerebral or systemic disease or condition known to cause cerebral cognitive dysfunction may be present. In the applicant’s experience, all patients who were classified as CDR.5 (“questionable dementia”) on the clinical dementia rating scale and who met these exclusions, also met the criteria for AACD.

Competitive ELISA for the detection and quantification of HO-1 is performed according to the procedure of wang et al. (1991) with the following modifications: immulon II ELISA plates (dynatech) are coated (50 ìl/well) with 0.5 ìg/ml HO-1 protein (dissolved in coating buffering containing na2co3 and nahco3 ph 9.6) and incubated overnight at 4° C. Excess protein is then washed off with washing buffer containing tris, nacl and tween-20.Brain anoxia wells are blocked with 3% bovine serum albumen (BSA) in coating buffer and incubated 2 hrs at 37° C. 75 ìl of plasma or solubilized membrane is then mixed with 75 ìl of HO-1 antibody (diluted 1/50,000) and incubated for 2 hrs. At room temperature. After blocking, wells are washed with 50 ìl/well of plasma or solubilized membrane and HO-1 antibody mixture is added and incubated overnight at 4° C. Any available antibody combines with the antigen exposed on the coated well. Secondary antibody consisting of alkaline phosphatase conjugated igg anti-rabbit (diluted 1/1000) in 0.1% BSA is added and incubated 2 hrs at 37° C. 50 ìl of the substrate (p-nitrophenyl phosphate disodium dissolved in diethanolamine ph 9.8) is added to each well and incubated for 20 minutes at room temperature.Brain anoxia the resulting colour reaction is read at 405 nm wavelength on a multi-well scanning spectrophotometer (molecular devices corp.). The standard curve consists of equal volumes of HO-1 protein (10, 5.0, 2.5, 1.0, 0.5, 0.1 ìg/ml) and HO-1 antibody (1/50,000) which are mixed and incubated for 2 hrs at room temperature. This mixture is then added to the protein coated, BSA blocked wells in the same manner as test samples. The results of % maximum inhibition are calculated based on the standard curve generated by the known amounts of HO-1 proteins in the standard samples.

EXAMPLE 2 •

Further analysis, however, suggests an alternative explanation. It was noted in the study that the most severely demented patients also seemed to be those who had deteriorated most rapidly.Brain anoxia A measure of rate of global deterioration was derived (the difference between two successive folstein mini-mental scores, multiplied by 3, divided by the intervening months). This formula was derived in view of the applicant’s past experience that the typical AD subject declines by 3 or 4 points on the MMSE per year. The association between rate of decline and HO-1 levels was the most robust of all (r=−0.72, p0.0001) (see FIG. 5). The implication is that the patients showing the most rapid decline demonstrated the lowest levels of HO-1. Accordingly, HO-1 serum levels offer the possibility of producing useful prognostic information; the presence of a low HO-1 level would imply more rapid deterioration over time in AD subjects.Brain anoxia the method can, accordingly, usefully be used to compare HO-1 levels in the same patient over time in order to prognosticate the disease.

EXAMPLE 3 •

Lymphocyte fractions were obtained by differential centrifugation of whole blood on ficol paque gradients as described above. Cytoplasmic RNA was isolated from the lymphocytes using an acid guanidinium thiocyanate-phenol-chloroform extraction method (chomezynski P et al., 1997). Six micrograms of RNA was denatured and size-separated by electrophoresis on 1% agarose/formaldehyde gels. RNA integrity was confirmed by ethidium bromide staining. The RNA was transferred onto hybond-N filter paper and covalently cross-linked to the membrane by UV light for two minutes.Brain anoxia the hybridization probe (HO-1; 1.0 kb) was prepared by random priming using the random primer DNA labelling system (feinberg A P et al., 1984). Prehybridization was performed for 12 hours at 42° C. In a buffer containing formamide deionized, 5×denhardt’s reagent, 6×SSPE and 0.5% SDS. The hybridization buffer consisted of the prehybridization buffer without 5×denhardt’s reagent, and 32P-labelled denatured DNA probe (noonberg S B et al., 1994). Equal loading of RNA was confirmed by hybridization with a cdna for the (housekeeping) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All washes were performed under stringent conditions (1×SSC and 0.2% SDS for 45 minutes at room temperature, 0.4×SSC and 0.2% SDS for 15 minutes at 65° C.).Brain anoxia the RNA hybridizing with cdna probes was visualized by autoradiography using an intensifying screen at −80° C. (church G M et al., 1984).

A kit or commercial package for carrying out the testing method includes a means for determining the concentration of heme oxygenase-1 (HO-1) and/or a nucleotide sequence encoding HO-1, in tissue obtained from a patient. It also includes instructions for comparing said concentration with an established standard of the corresponding concentration of HO-1 and/or an HO-1 encoding nucleotide sequence in corresponding tissue obtained from at least one normal elderly control person or from the patient at another time.

TABLE 1

AGE

SEX

MMSE

EDUC.

DIAGNOSIS

II

(mean)

F/M

(mean)

brain anoxia

(mean)

CONTROL

31

62.5

17/14

29.4

14.1

Age 40-60(NYC)⊕

7

47.1

1/6

30

16.6

60 +(NBC) *

24

77.9

16/8

28.9

11.7

AACD *

25

76.3

15/10

26.9

10.5

AD *

50

76.8

27/23

21.1

10.9

NAD *♦

16

74.3

6/9

11.5

LBD

4

71.25

2/2

18.2

CBGD

2

69.6

1/1

7.5

HD

1

63

M

24

NPH

2

81.0

2M

25

MID

4

77.5

1/3

6.2

MSA

1

86

M

11

DOWN

1

55

F

0

HT

1

86

F

0

PD 

21

68.7

10/11

29.0

ALS Δ

14

55.7

8/6

28.9

SALS

13

56.1

8/5

28.8

FALS

1

50

M

30

STR #

5

61.8

1/4

26

RD