Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-22
2002-04-23
Kemmerer, Elizabeth (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S012000, C436S501000, C530S350000, C530S399000
Reexamination Certificate
active
06376197
ABSTRACT:
SPONSORSHIP
Support for the research disclosed herein was provided by the University of Maryland, Baltimore and the Interstitial Cystitis Association.
FIELD OF THE INVENTION
The field of this invention specifically relates to the diagnosis of Interstitial Cystitis (IC) by measuring levels of HB-EGF in the urine of IC patients.
BACKGROUND OF THE INVENTION
Interstitial cystitis (IC) is a chronic inflammatory disease of the bladder for which the etiology is unknown. IC often has a rapid onset with pain, urgency and frequency of urination, and cystoscopic abnormalities including petechial hemorrhages (glomerulations) or ulcers that extend into the lamina propria (Hunner's ulcers)
1,2
. The rapid onset of IC is followed by a chronic course with partial remissions and re-exacerbations, which can continue for up to 30 years
1,2
. As a result of the absence of a specific cause for and lack of understanding of its pathogenesis, there is currently no generally accepted treatment proven to be reliably efficacious.
Various groups have studied IC and speculated as to its cause. Proposed etiologies include infection, allergic or immune disorders, endocrinologic disturbance, toxic urinary chemicals, defective transitional mucosa, psychiatric disorders, neurogenic disorders, lymphatic obstruction, or vascular obstruction. Proposed treatments include pentosan polysulfate, anti-inflammatory or immunosuppressant therapy, muscle relaxants, anti-histamines, and analgesics. Of these, only pentosan polysulfate has been approved by the FDA. However, none of the proposed therapies, including pentosan polysulfate, is universally accepted or universally efficacious. As a result, there is a long felt need for adequate therapy of this poorly understood and frequently misdiagnosed disorder.
Certain morphologic and histologic features of IC suggest that the epithelium is usually abnormal in this disease
3-5
, with evidence for changes in the bladder mucin layer
6
, denudation or thinning of the bladder epithelium and rupture of the mucosa
3-5,7
, and intraurothelial infiltration of urinary proteins such as Tamm-Horsfall protein
8
. In addition, activation of bladder epithelial cells appears to be abnormal in IC, with altered expression of specific cellular proteins
9
. These changes coupled with the chronic nature of IC suggests the possibility of impaired regeneration of normal bladder epithelium. In previous experiments, we discovered a 1-3 kDa peptide in the urine of IC patients that inhibits the proliferation of cultured normal adult human bladder epithelial cells, suggesting that it may be related to the pathogenesis of this disorder (see co-pending application Ser. No. 08/944,202 now U.S. Pat. No. 5,962,645). This peptide is hereafter referred to as the anti-proliferative factor or APF.
The uninjured postnatal urothelium regenerates very slowly, but rapid proliferation of uroepithelial cells in vivo can occur during tissue regeneration in response to injury
10
. The limited data that exist for bladder epithelial cells suggest their replication and differentiation are probably influenced by specific paracrine or autocrine growth factors and their regulatory proteins, similar to other types of epithelial cells
10-21
. Epithelial cell growth factors known to be present in normal human urine include epidermal growth factor (EGF), insulin-like growth factors (IGF's), insulin-like growth factor binding proteins (IGFBP's), heparin-binding epidermal growth factor-like growth factor (HB-EGF), platelet-derived growth factors (PDGF-A and B), fibroblast growth factors (FGF1 and 2), and transforming growth factor beta (TGF&bgr;). EGF, which is produced primarily by cells in the thick ascending limb of Henle and the distal convoluted tubule
22
, is present in high concentrations in urine, and can stimulate, but is not required for, mouse bladder epithelial cell proliferation in vitro
23
. IGF1 and IGF2 are produced by both kidney and bladder cells and appear to be required for bladder epithelial cell proliferation
10,21,24
. The major IGFBP's found in human urine, which can regulate the activity of IGF1 and 2 are IGFBP-2 and IGFBP-3
25
. HB-EGF is also known to be produced by human bladder epithelial cells and can stimulate their growth in vitro
26,27
. In contrast, current data suggest that the PDGF's, FGF's and TGF&bgr; affect bladder epithelial cell migration and/or differentiation, but their role in cell proliferation remains uncertain
10,21
.
Exogenously applied growth factors can stimulate epithelial wound repair
11-13
. Since IC is histologically characterized by epithelial abnormalities and because the mucosal defects present in IC result in exposure of basal undifferentiated cells and their growth factor receptors to urine growth factors, we reasoned that abnormally low levels of urinary growth factors, such as HB-EGF, that stimulate bladder epithelial cell proliferation could adversely affect bladder epithelial wound repair and be part of the etiology of IC.
We measured urinary levels of HB-EGF in women with IC, asymptomatic control women without bladder disease, and women with acute, self-limited bladder epithelial damage from bacterial cystitis. We discovered that urine levels of HB-EGF are specifically and significantly decreased in the urine of IC patients.
Based on the above, we have concluded that urine levels of HB-EGF may be used for diagnosing Interstitial Cystitis (IC).
SUMMARY OF THE INVENTION
Human bladder epithelial cells are known to produce HB-EGF
26
. Since IC is histologically characterized by epithelial abnormalities and because the mucosal defects present in IC result in exposure of basal undifferentiated cells and their growth factor receptors to urine growth factors, we reasoned that abnormally low levels of urinary growth factors, such as HB-EGF, that stimulate bladder epithelial cell proliferation could adversely affect bladder epithelial wound repair and be part of the etiology of IC.
It is the object of the invention to provide a reliably effective diagnostic for diseases associated with inhibited epithelial cell proliferation, particularly bladder epithelial cell proliferation, more particularly interstitial cystitis (IC), using heparin-binding epidermal growth factor-like growth factor (HB-EGF) which is capable of inhibiting the anti-proliferative activity present in most IC urine specimens. Our findings indicate that complex changes in the levels of urine growth factors are associated with IC, including significant and specific decreases in BB-EGF levels in the urine of IC patients.
REFERENCES:
patent: 5811393 (1998-09-01), Klagsburn et al.
patent: 5962645 (1999-10-01), Keay et al.
Freeman, M et al. Heparin-binding EGF-like Growth Factor is an Autocrine Growth Factor for Human Urothelial Cells and is Synthesized by Epithelial and Smooth Muscle Cells in the Human Bladder. Journal Clinical Investigation vol. 99 (5) 1028-1036 (1997).*
Hanno, P.M., et al. eds.Interstitial cystitis.London: Springer-Verlag (1990).
Held, P.J. et al. “Epidemiology of interstitial cystitis: 2.”Interstitial cystitis.: 29-48. London: Springer-Verlag (1990).
Johansson, S.L. et al. “Clinical feature and spectrum of light microscopic changes in interstitial cystitis.” J. Urol, 143: 1118 (1990).
Oravisto, K.J. et al. “Interstitial cystitis: Clinical and immunological findings.” Scand. J. Urol. Nephrol. 4:37 (1970).
Skoluda, et al. “Kritische Bemerkungen zur Immunopathogenese der Interstitiellen Cystitis.” Urologe, 13: 15 (1974).
Parsons, et al. “Epithelial dysfunction in nonbacterial cystitis (interstitial cystitis).” J. Urol 145:732 (1991).
Smith, B.H. et al. “Chronic ulcerating interstitial cystitis (Hunner's ulcer).” Arch. Path. 93:76 (1972).
Fowler J. Jr., et al. “Interstitial cystitis is associated with intraurothelial Tamm-Horsfall protein.” J. Urol. 140: 1385 (1988).
Liebert, M., et al. “Evidence for urothelial cell activation in interstitial cystitis.” J. Urol. 149: 470 (1993).
deBoer, W.I., et al. “Expression of growth factors and receptors during specific phases
Hise Michael K.
Keay Susan K.
Warren John W.
DeBerry Regina M.
Fuierer Marianne
Hulquist Steven J.
Kemmerer Elizabeth
University of Maryland Baltimore
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