CA2289690A1 - Method for assessing wound healing - Google Patents

Abstract

The present invention is directed to a method for assessing the healing status of a wound in a mammal, which method comprises measuring the amount of at least one lymphocyte cell membrane antigen present in a sample of body fluid or tissue. It has been found that the ratio of T-lymphocyte antigens CD4 and CD8 vary depending on the healing status of a wound. Assessment of the healing status assists in identifying the correct treatment to promote healing.

Description

Method for Assessing Wound Healing The present invention relates to a method for assessing the healing ;status of a wound in a mammal. In particular it relates to a method for determining whether healing of a wound is taking place or is likely to take place by measuring t:he amount of at least one lymphocyte cell membrane antigen present in a sample of body fluid or tissue.
When a wound occurs the upper layer of skin, i.e. the epidermis, which comprises keratinocytes, is punctured, so too is the layer below the epidermis, i.e. the dermis which comprises mainly fibroblast cells and other cell types.
Following wounding, an inflammatory response occurs which involves lymphocytes migrating to the site of injury, or "wound bed".
Lymphocyte cell or other cell surface or membrane antigens have been characterized using monoclonal antibodies and assigned. a CD (cluster of differentiation) number. For example, CD:~ is a protein complex associated with the T-lymphocyte receptor, and CD4 and CD8 are glycoprotein adhesion mo7_ecules present on T-lymphocytes. CD25 is the receptor fo:r interleukin-2. CD19 and CD20 are membrane antigens found on H-lymphocytes.
T-lymphocytes are divided into at least three different types or subsets; cytotoxic T cells (T~), suppressor T cells (TS) and helper T cells (TH) . TH cells generally possess the cell surface: marker CD4 whilst TS cells carry CD8. Several of the cell surface markers that are present on T-lymphocytes have been found in soluble form in, for example, plasma and serum.
Simon M. et al in J Derm., 23, 305-309, 1996, examined the expression of the thrombospondin receptor (CD36) by keratinocytes in acute uninflamed wounds.
Greiling et al developed an in-vitro assay to assess the role of the glycoprotein CD44, which is expressed by human dermal fibroblastsin fibroblast migration during wound repair (see Annual Meeting of 6th International Congress on Cell Biology, San Francisco, U.S.A., Dec. 7-11 1996.
Molecular Biology of the Cell 7 (Suppl.) 1996.
Mertz, P.M. et al in J. Invest. Dermatol., 98(4), 634, 1992 examined the expression of CD44 by keratinocytes during burn wound healing.
W097/11369 describes a method for quantifying an inflammatory response by measuring the cellular infiltration in a tissue biopsy of a warm-blooded animal.
W096/25670 relates to a cell enumeration immunoassay for quantising the number of cells in a subpopulation of a cell sample. The assay is described as an efficient alternative to flow cytometry.
U.S. Patent No. 5 006 459 is directed to the measurement of soluble T-lymphocyte cell differentiation antigens and the use of such measurements in the diagnosis and therapy of diseases and disorders. In specific embodiments it describes the measurement of serum or plasma interleukin-2 receptor levels to detect leukaemia or lymphoma, as. well as the measurement of CD8 levels for the differential diagnosis of renal allograft rejection, as distinct from Cyclosporin A nephrotoxicity, and of rheumatoid arthritis, as distinct from other joint diseases.
U. S . Pa.tent No . 5 292 636 is directed to methods' for the measurement of soluble CD4 antigens, which measurements can be used to diagnose a state of immune activation, to diagnose rheumatoid arthritis, to monitor therapeutic efficacy (e. g. of AIDS treatment) or to stage adult T cell leukaemia.
U.S. Patent No. 5 426 029 describes the measurement of soluble leuc:ocyte surface markers, soluble T cell growth factor receptors, soluble complement receptors, soluble T
cell differentiation antigens and the use of such measurements in the diagnosis or therapy of diseases and disorders. In particular it is directed to the measurement of soluble CD35 which can then be used in the diagnosis of autoimmune disease such as systemic lupus erythematosus, rheumatoid arthritis, glomerulonephritis, inflammation, infectious disease such as AIDS, transplantation, blood transfusion, haemodialysis, cardiopulmonary bypass thermal injury, adult respiratory distress, sepsis and barotrauma.
Following tissue injury, an ordered sequence of cellular events is initiated that in healthy subjects leads to wound closure. These events can be divided into the three phases of an initial inflammatory phase, followed by a second phase of proliferation and a final phase of matrix formation and remodelling [Clark, R.A.F: Wound Repair:

Overview and general considerations. In: The Molecular and Cellular Biology of Wound Repair (RAF Clark, ed), 2nd edn., Plenum Press, NY and London, 1996; 3-50]. This process is complex and many of the events may occur in parallel through each phase. The acute inflammatory phase is characterised by a neutrophil infiltrate which is rapidly replaced by mononuclear cells [Dyson M., Young SR, Pendle CL, et al.
Comparison of the effects of moist and dry conditions on dermal repair. J. Inv Derm 1988; 91: 435-9] with the monocyte component maturing to form wound macrophages.
These cells persist through the following phases of healing and the macrophage, in particular, is considered to play a key role in regulating the events that lead to successful wound healing.
Based on data obtained from experimental animal models, it has been postulated that cells of the immune system, such as lymphocytes and macrophages play a central role in normal wound healing. Depletion of T-lymphocytes from normal mice by in vivo administration of a T-lymphocyte specific monoclonal antibody impairs the healing of incisional wounds.
Depletion of the T-helper subset had no inhibitory effect on healing whilst depletion of the T-suppressor subset enhanced the healing rate. This evidence suggests that one role played by T-lymphocytes at the wound site is a negative one exerted by suppressor cells. This hypothesis is supported by the observation that CD8' cells present in non-healing wounds within tumours have been demonstrated to inhibit fibroblast proliferation.
Little direct evidence is available to define the positive, oz- negative, functional role of leucocytes in human wound healing. Both T-lymphocytes and macrophages are 5 associated with healing surgical wounds. This leucocyte infiltration gradually decreases with time after wounding except in hypertrophic and keloid scars where high numbers of T-helper cells were observed suggesting that the presence of this population may generate a positive signal for fibroblast proliferation.
These data suggest that lymphocytes may play a role in providing a positive signal for healing to proceed and that as the healing process progresses toward completion it may be switched off in part as a consequence of a counter-regulatory signal provided by suppressor lymphocytes.
Although some work has been carried out to suggest that T-lymphocytes do play an active role in skin healing (Barbul A: Immune aspects of wound repair. Clin. Plast. Surg. 17:33-442, 1990; Adolph VR. DiSanto SK, Bleacher JC, et al. The potential role of the lymphocyte in fetal wound healing. J
Paediatric Sur 28:1316-20, 1993; Wojciak B, Crossan JF. The effects of T cells and their products on the in vitro healing of epitenon cell microwounds. Immunology 83: 83-98, 1994) most lzas been done on animal or in vitro models.
Martin CW and Muir IF: The role of lymphocytes in wound healing. Br J Plast Surg. 43: 655-62 is the only reference to present evidence that they may play such a role in human tissue.

WO 98/54575 i'CT/GB98/OI520 U.S. Patent No. 5 270 168 describes a method for the diagnosis of non-healing ulcers in humans by assaying for certain cell adhesion-related proteins, such as fibronectin and vitronectin, or their degradation products in ulcer exudate.
It has now been found that by measuring the amount of certain lymphocyte cell membrane antigens present in a sample of body fluid or tissue the healing status of a wound can be assessed. Furthermore, by monitoring the amount of the cell membrane antigen over a period of time, the clinician can determine the healing status of a wound that is whether the wound is healing or likely to heal, or whether the wound has become a chronic wound which is not healing.
According to one aspect of the invention there is provided a method for assessing the healing status of a wound in a mammal comprising measuring or detecting the amount of at least one lymphocyte cell membrane antigen present in a sample of body fluid or tissue.
Preferably the method comprises measuring the amount of at least one lymphocyte cell membrane antigen from each of two different types or subsets of T-lymphocytes and assessing the healing status of the wound by comparing the relative proportion of the two types or subsets of T
lymphocytes.
Thus, for example, the method may be used to measure firstly the proportion of T-suppressor lymphocytes present in a sample by detecting the amount of CD8 antigen, and secondly the proportion of T-helper lymphocytes present in a sample by detecting the amount of CD4 antigen. The healing status can then be determined by the ratio of CD4:CD8.
The sample of body fluid may be blood or serum obtained by venupuncture. Alternatively, the body fluid may be wound fluid which is present on the surface of the wound, and which rnay be sampled by soaking it into an absorbent material such as filter paper. The wound fluid or exudate is then eluted from the absorbent material prior to measuring the amount of lymphocyte cell membrane antigen.
Alternatively, the wound fluid present at the surface of a wound can be sampled by aspiration with a syringe and needle or pipette.
The sample of body tissue may be a sample of wound tissue or tissue peripheral to the wound, which may be obtained by :biopsy.
In order to identify and quantify the lymphocytic infiltration of the biopsied tissue, serial sections can be stained using immunohistochemical staining with a panel of anti-lymphocytic monoclonal antibodies. The types of lymphocytes and proportions of subpopulations can then be compared.
Alternatively, where the sample to be assessed is a fluid, such as wound fluid, the types of lymphocytes and proportions of subpopulations may be determined using an immunological assay such as an enzyme-linked immunosorbent assay (ELISA). ELISA assays are commercially available for soluble lymphocyte receptors including CD4, CD8 and CD25.
An ELISA for the soluble B lymphocyte associated receptor CD23 is also available. Other tests suitable for determining the type and proportion of lymphocytes present S in a fluid sample include radioimmunoassays, precipitin reactions, gel diffusion reactions, immuno-diffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays.
Thus, in order to determine, for example, the proportion of soluble CD4 antigen present in a fluid sample, the sample may be reacted with a first antibody which specifically binds to CD4 or its degradation products.
After treatment with the first antibody, the sample may be reacted with a second antibody specifically binding the first antibody. The second antibody is conjugated to a label such as an enzyme, peroxidase for example, which forms a chromophoric product on contact with a substrate for the enzyme. The chromophoric product can then be quantified by reading the optical density at the appropriate wavelength.
In one embodiment, the method for assessing the healing status of a wound in a mammal comprises monitoring lymphocyte populations within a wound by sampling fluid from the wound, detecting or measuring soluble CD4 T-helper lymphocyte antigen in the sample, detecting or measuring soluble CD8 T-suppressor lymphocyte antigen in the sample, and determining the presence or absence of healing of the wound by the relative proportion of CD4:CD8.

In another embodiment the method for assessing the healing stat:us of a wound in a mammal comprises monitoring lymphocyte populations within a wound by obtaining a sample of tissue from the wound, detecting or measuring the proportion of T-helper lymphocytes in the sample, detecting or measuring the proportion of T-suppressor lymphocytes in the sample, and determining the presence or absence of healing of the wound by the relative proportion of T-helper lymphocytes to T-suppressor lymphocytes.
Using t;he method of the present invention it has been found that early in healing the CD4:CD8 ratio is high, that is in the range of 3.0 to 8Ø As the wound closes, so the CD4:CD8 ratio falls within a range of 0.5 to 2.5.
Concomitantly the expression of CD25 antigen increases as the wound heals. Thus, for example, the amount of CD25 may be about !3% shortly after wounding, rising to 18%
immediately prior to wound closure.
In another embodiment the method of the invention may be used to measure or detect the amount of B lymphocytes in a sample from a wound, by measuring the amount of CD19 or CD20.
Using this method it has been found that initial levels of B lymphocytes shortly after surgery are the same as that found in non-healing chronic wounds. As the wound heals, so the level of B lymphocytes rises.
In yet ~~ further embodiment the method of the invention may be used to measure or detect the amount of CD3 or CD25 lymphocyte cell membrane antigen.

Using the method of the invention it has been found that:
1. a high CD4:CD8 ratio occurs early in wound healing.
2. a low CD4:CD8 ratio occurs in non-healing chronic 5 wounds.
3. the CD4:CD8 ratio is elevated prior to healing of a previously non-healing wound.
4. high amounts of CD25 occur in healing wounds prior to closure.

10 5. low amounts of CD25 occur in non-healing chronic wounds.
6. high amounts of B lymphocytes occur in healing wounds prior to closure.
7. low amounts of B lymphocytes in non-healing chronic wounds.
According to a further aspect of the invention there is provided a wound healing assessment kit for carrying out the method of the invention which comprises a first antibody that binds to a lymphocyte cell membrane antigen and a labelled antibody that binds to the first antibody.
Preferably the kit also contains a second antibody that binds to a different lymphocyte cell membrane antigen and a labelled antibody that binds to the second antibody.
The first antibody in the kit binds to the CD4 cell membrane antigen from T-helper lymphocytes, whilst the second antibody in the kit binds to the CD8 cell membrane antigen from T-supressor lymphocytes.
The method of the invention will aid in the assessment of the effect of wound treatment, both experimental and routine, and. in the assessment of patients at presentation.
Using this method allows the clinician to obtain an early indication of healing which will then assist them to evaluate different wound dressings and select the best~type to promote a.nd achieve complete healing.
Description of the drawings Figure 1 shows the proportion of B lymphocytes in chronic and acute wounds.
Figure 2 shows the decline in CD4:CD8 ratio in an acute wound as it heals.
Figure 3 shows the ratio of CD4:CD8 in chronic and acute wounds.
The invention will be described further with reference to the following examples.
Example 1 Study of T-lymphocytes within chronic lea ulcers Materials anal Methods A 6mm punch biopsy was taken under local anaesthesia from the margin of each patient's leg ulcer. Twelve patients were selected with chronic leg ulcers due to venous disease or whose general condition, immobility and leg oedema prevented healing. These wounds had been present for a minimum of 6 months and clinical records showed no evidence of healing occurring in the 6 weeks prior to biopsy.
Biopsies were snap frozen in liquid nitrogen and 6~e cryostat sections mounted on poly-L-lysine treated microscope slides. Slides were stored desiccated at -20°C
for up to 14 days prior to staining. Serial sections. were fixed in dry acetone, washed in phosphate buffered saline (PBS) 3 times and incubated in optimal dilutions of monoclonal antibody (MAb) for 30 minutes. The antibody panel, with antigen specificity and working dilutions in parentheses, was applied to serial sections in the following order: anti-CD45 (all leucocytes, 1:20), anti-CD19 (B-lymphocytes, 1:25), anti-CD3 (T-lymphocytes, 1:50), anti-CD4 (T-helper/inducer lymphocytes, 1:10), anti-CD8 (T-suppressor/cytotoxic lymphocytes, 1:30), anti-CD25 (interleukin-2 receptor expressed on activated T-lymphocytes and macrophages, 1:20), anti-CD45RA (suppressor/inducer T-lymphocytes and B-lymphocytes, 1:20), anti-CD45R0 (Memory T-lymphocytes, 1:50), anti-CD68 (macrophages, monocytes, 1:40), anti-CD14 (LPS receptor on macrophages, monocytes and neutrophils, 1:30), anti-CD16 (Fc~ylll receptor on activated macrophages, neutrophils and NK cells, 1:30), anti-CD35 (C3b receptor on macrophages, monocytes, B-lymphocytes and neutrophils, 1:30) and anti-HLA Class 11 antigen (1:100).
They were then washed 3 times in PBS and antibody localisation identified by a standard streptavidin-biotin peroxidase technique (Vector Laboratories, Peterborough, UK) with final reaction product developed using 3,3'-diaminobenzidine (DAB). The sections were counterstained with Ehrlich's haemotoxylin, dehydrated, cleared and mounted in DPX mounting medium. Positive staining was seen as a brown-black deposit and non-stained cells could be clearly distinguished as blue counterstained nucleated cells with no associated :brown DAB stain.
All monoclonal antibodies (MAbs) used were obtained from Dako Lt: d, High Wycombe, UK except for 2H4 (anti-CD45RA) which was obtained from Coulter Immunology Division, Hialeah, F1., USA.
For each biopsy the area underlying the epidermis adjacent to the wound margin was identified. Commencing at the epidermal tip and moving distally to the wound using a x20 objective six adjacent fields were counted in the papillary dermis along with the six underlying fields immediately below. This counting method included the area of dermis at the wound margin that contained the highest leucocyte density demonstrated by anti-CD45 staining. The numbers of positive stained cells were counted per field and the average number of stained cells/field calculated.
Manual counting with a microscope eyepiece grid was used as it was not possible to enumerate the stained cells using a computerised image analysis system (IBAS) because of the asymmetric distribution of stained cells and the close approximation of stained cell membranes within aggregates particularly in the perivascular regions.
Positive cells were enumerated in the same way in corresponding areas of each subsequent serial section stained with the MAb panel. The CD4:CD8 ratio was calculated by dividing the average number of CD4' cells/field by the average number of CD8' cells/field and expression of other antigens by use of the following formulae;
i) ~CD25' T-lymphocytes - CD25' cells/field x 100;
CD3' cells/field ii) ~B-lymphocytes - CD19' cells/field x 100 (CD3'cells/field) + (CD19'cells/field) iii} ~CD16(35) macrophages = CD16(35)' cells/field x 100;
CD68' cells/field RESULTS
With respect to leucocytes infiltrating the wound margin sections stained with the pan leucocyte marker CD45 allowed the tissue to be divided into four areas. These were (i) the epidermis which by comparison to normal skin was thickened both at the immediate wound edge and also in adjacent areas distal to the wound, (ii) an area including the papillary dermis and the upper region of the reticular dermis which could be delineated by the high numbers of blood vessels and associated leucocytes close to the wound margin, (iii) the reticular dermis distal to the wound margin characterised by the absence or low density of blood vessels and few leucocytes and (iv) associated wound bed tissue.
Some of the antigens under study, CD4, CD25 and CD45R0, may be expressed by macrophages. Staining for these antigens appeared either as a membrane type staining pattern on cells of a small round morphology which were assumed to be lymphocytes or as a cytoplasmic more diffuse staining pattern where the antigen was expressed on larger cells of 5 asymmetric nnorphology. The latter staining pattern. was identical to that found in sections stained for the macrophage associated CD68 antigen and these cells were therefore considered to be macrophages. These two morphologies could be distinguished microscopically and only 10 those cells exhibiting a lymphocytic morphology were enumerated for expression of CD4, CD25 and CD45R0.
Leucocyte populations present within the infiltrate adjacent to the wound margin were enumerated. Cells of a macrophage morphology were distributed in the intervascular 15 areas of the papillary dermis and throughout the reticular dermis in close approximation to non-stained cells of fibroblast morphology.
Cells of lymphocytic morphology, small round cells with little cytoplasm and a typically round nucleus, were identified in greatest numbers in perivascular areas.
Additional CD45' cells were identified throughout the epidermis adjacent and distal to the wound margin. These cells had a dendritic morphology and were CD3-/CD68-/HLA
Class 11' indicating that they were typical Langerhans cellslo [Weber-Matthiesen K, & Wolfram S. Organisation of the Monocyte/Macrophage system of normal human skin. J Invest Dermatol 1990; 95: 83-B9]. Significant numbers of CD45' cells were also identified within the wound bed tissue but it was not possible to enumerate these with any degree of accuracy because of non cell associated background staining in this area.
The wound margin leucocyte population was comprised essentially of CD3' T-lymphocytes and CD68' monocytes and macrophages.
Lymphocyte Antigen Expression The perivascular infiltrate in the biopsies examined contained a significant number of CD3' T-lymphocytes. Few CD3' cells were found in tissue remote from vessels and only isolated CD3' cells were observed within the epidermis.
In the majority of chronic wounds tested the ratio of CD4':CD8' lymphocytes was within the range 0.5 to 2.2 (mean - 1.5~0.6)(Table 1).

TABLE 1 Lytnphocyte antigen expression at the margin of Chronic Wounds Patient No. %8-lymphocytesCD4:CD8 %CD25' T Lymphocytes 1 0 1.7 6 5 2.2 2 4 4 1.8 5 5 0 1.4 NT

6 6 0.5 2 7 0 1.6 5 8 2 2.0 6 9 0 0.7 7 NT - Not tested Stained cells were counted in serial sections in the same area of each section adjacent to the wound margin approximating to the area of leucocyte infiltrate. Total CD68' macrophages were counted followed by cells expressing each antigen in subsequent sections. The data shown indicates the percentage of total macrophages expressing each individual antigen.
To characterise the T-lymphocyte population further their expression of CD45RA and CD45R0 antigen was also determined. To eliminate interference by CD45RA expressing B-lymphocyteFi only biopsies which contained Lew (<4~) B-lymphocytes were evaluated for expression of CD45 antigen isotypes. CD45R0' T-lymphocytes predominated in the 4 biopsies examined (Table 2) with the proportion varying from 62~ to 94~. The majority of positive cells were located within the T-lymphocyte populations in the perivascular areas of the biopsies.
TABLE 2 Expression of CD45RA and CD45R0 by T-lymphocvtes at ."~,-.-.;,-, of r-hrnni c~ wounds Patient No. %CD3+ loCD45RA' %CD45R0' 4 g6 26 43 o 11 gg 14 92 NT - Not tested Stained cells were counted in serial sections in the same area of each section adjacent to the wound margin approximating to the area of leucocyte infiltrate. Total lymphocyte counts were derived by summation of the counts of CD3' T lymphocytes and CD19' B lymphocytes in adjacent sections. B lymphocyte data are expressed as a percentage of this total lymphocyte count. CD25' lymphocytes are expressed as a percentage of the total CD3' lymphocyte count only.
Macrophage antigen expression CD68' cells were distributed throughout the entire dermis with particularly dense aggregations of positive cells in the: perivascular areas. These compromised either smaller round cells, presumably recently emigrated monocytes, and larger elongated cells which formed a perivascular sheath. CD68' positive macrophages were also identified within the intervascular areas of the dermis.
Processes extending from these cells interdigitated with non-stained cells.
In all biopsies evaluated the expression of CD16 and CD35 antigen was essentially identical with both being elevated in 4/10 wounds tested and the remaining wounds demonstrating little expression (<12~ of cells positive)(Table 3). In all the biopsies except one (Patient 10) the greatest number of cells stained for these antigens was restricted to those cells within the perivascular areas .
In the one biopsy (Patient 10) where the majority of macrophages were positive for these two antigens clear staining of cells throughout the dermis was observed although stronger staining was observed in perivascular areas.
In contrast to CD16 and CD35, CD14 antigen expression was more uniformly distributed throughout the dermis.
Significant numbers of CD 14' cells were identified within all biopsies and in only one biopsy (Patient 1) did markedly less than 50~~ of macrophages express the CD14 antigen.

TABLE 3 Macrophage antigen expression at the mar4in of chronic wounds Patient No. CD16+ CD35' CD14' as 96 of CD68' cells NT - Not tested 15 Stained cells were counted in serial sections in the same area of each section adjacent to the wound margin approximating to the area of leucocyte infiltrate. Total CD68' macrophages were counted followed by cells expressing each antigen in subsequent sections. The data shown 20 indicates the percentage of total macrophages expressing each individual antigen.

Example 2 Studv of change in lymphocyte subpopulations associated with human wound healin4 Materials and Methods Biopsies 6mm punch biopsies were obtained under 1% lignocaine local anaestlnetic from the edge of surgically excised human pilonidal sinus excision sites. Rates of healing of the wounds were evaluated using the method of Marks J, Hughs LE
& Harding KG. Prediction of healing time as an aid to the management o:E open granulating wounds. World J Surgery 7:
64I-645 (1983) Serial biopsies were taken from twelve patients at day 0 (within 5 days of surgery) day 7, day 21, and day 42 if the wound had not healed. In comparison, single biopsies were taken from the wound edge of 10 chronic venous leg u~'.cers .
Processina oi: biopsies Biopsies were snap frozen in liquid nitrogen and 6~m cryostat sections mounted on poly-L-lysine treated microscope slides. Slides were stored desiccated at -20°C
for up to 14 days prior to staining. Serial sections were fixed in dry acetone, washed in phosphate buffered saline (PBS) 3 times and incubated with monoclonal antibody (MAB) (Dako Ltd, High Wycombe, UK) for 30 minutes. The antibody panel and specificities are described in Table 3 and were applied to serial sections in the order shown. They were then washed 3 times in PBS and antibody localisation identified by a standard streptavidin-biotin peroxidase technique (Vector Laboratories, Peterborough, UK) with final reaction product developed using 3,3'-diaminobenzidine (DAB). The sections were counterstained with Ehrlich's Haematoxylin, dehydrated, cleared and mounted in DPX
mounting medium.
Identification and interpretation of lymt~hocytic infiltrate The lymphocytic infiltrate of all biopsies was quantified by counting X40 magnification views. Significant numbers of positively staining cells were noted within the wound bed, but it was impossible to quantify these accurately because of the great amount of background staining and particulate manner which also stained in this region. The leucocyte infiltrate was therefore examined by counting numbers of positive cells in an orderly manner consisting of 6 fields commencing directly under the migrating epithelial tip and progressing distally in both upper and lower dermis achieving 12 fields in total for each biopsy. Subpopulations are expressed in the following way: B-lymphocytes as proportion of total lymphocytes (B and T lymphocytes), T-lymphocytes as proportion of total lymphocytes, CD27' as ~ of CD3' T lymphocytes, CD25' as ~ of CD3' T lymphocytes, and CD4':CDS' T lymphocytes are expressed as a ratio (Table 2).
RESULTS
Healing of pilonidal sinus excisional wounds progressed over a pe~_-iod of seven weeks. CD45 staining, which identified all leucocytes was generally concentrated under the migrating epithelial tip and towards the wound edge.
Cells of lymphocytic morphology, small round cells with little cytoplasm and a typically round nucleus, were found preferentially in the perivascular areas. Macrophages were found diffusely distributed within the dermis with no distinct pattern of organisation.Further analysis of the lymphocyte population with subset specific MAbs showed statistically significant changes as healing of the wounds progressed.
B Lymphoc r~te~s B-lymphocytes were identified in greatest numbers at the wound edge, but were also congregated in small clusters within the upper and lower dermis. There was a consistently low proportion of B lymphocytes of 2.9(~1.3)% in chronic wounds. In the acute wounds, there was a significant rise in the proportion of CD19' cells as healing progressed from 3.7 (~I.2) % t:o 17.7 (~4.1) % at day 7, 21. 1 (~4 .0) % at day 21, and 27.2 (~4.2) % at day 42. The difference between day 0 and day 7 was significant to a level of p=0.016, and the difference between day 0 and biopsies taken on days 21 and 42 significant to levels of p=0.0014 and p<0.001 respectively (Fig 1). To confirm that the cells identified as CD19' were B lymphocytes, sections were stained Lor the separate B :Lymphocyte associated antigen CD20. Expression of CD20 correlated well with CD19 staining, both spatially WO 98!54575 PCT/GB98/01520 and numerically (r~=0.94, P<O.OOl,n=17).
T Lvmphocytes:
As with B lymphocytes, T lymphocytes were identified in their greatest numbers directly at the wound edge, but preferentially in the perivascular areas.
CD4:CD8 ratio:
In acute wounds, the CD4:CD8 ratio initially observed within the T lymphocyte population at the wound margin was 4.0(~0.5). As healing progressed, the CD4:CD8 ratio significantly decreased so that prior to wound closure it was 1.3(~0.7-1.9) (p<0.01) (Fig 2). This was a result of an increase in the absolute numbers of CD8' lymphocytes from 6.7(~0.9) to 11.6(~1.7) per/field (p=0.041), and a decrease in the number of CD4' lymphocytes from 22.6(~3.2) to 16.15(~2.5) per/field (p=0.045). When data for all biopsies taken throughout the healing process was pooled, there was an overall ratio of 3.0(~0.3). In chronic wounds there was a consistently low CD4:CD8 ratio of 1.9(~0.6)(fig. 3)). This was significantly lower than that of the day 0 acute wounds (p=0.016) .
CD27' & CD25' T lymphocytes The lymphocyte associated expression of the CD25 antigen (Interleukin-2 [IL-2] receptor) was generally lower than CD27. However, expression of both antigens increased as normal healing progressed. Numbers of CD25' cells increased from 9.4(~1.0)% of T lymphocytes at day 0 to 17.9(~3.5)% at day 42 (p=0.056). The corresponding increase in numbers of CD27' lymphocytes was from 26.2 (~4.0) % to 46. 9 (~6.0) S (p=0.046).
Conclusion A high CD4:CD8 was found in the initial stages of wound healing whi~~h declines as healing progresses. Such a decline is the result of both a significant rise in the 10 number of T~; lymphocytes and a decline in the number of Th lymphocytes. This proliferation of CD8" Ts lymphocytes is consistent with an increase in expression of CD25 and CD27, which are markers of lymphocyte activation and proliferation. The low levels of potentially 15 'downregulatory' Ts cells in the initial stages of healing may therefore represent the initial attempt of the wounded tissue to close the acute defect . The high levels in the final stages of normal healing may contribute to the 'switching off' of this process. This would also be 20 consistent with the findings in chronic wound tissue: where a consistently low CD4:CD8 ratio is seen in the non-healing wounds, reflecting high levels of these 'downregulatory' Ts lymphocytes, whose presence may contribute to the chronicity of the wound.

25 It was a surprising finding that CD19" B-lymphocytes comprised a large proportion of the lymphocytic infiltrate of normally healing wounds. In addition, they were significantly more numerous in acute wounds when compared to those which were chronic and non-healing.
TABLE 3 Immunocytochemistry Primary Monoclonal Antibody Panel Antigen: Cellular Distribution:

CD45 Allleucocytes CD19 B lymphocytes CD20 B lymphocytes CD3 T lymphocytes CD4 T hefperlinducer lymphocytes l0 CD8 T suppressorfcytotoxic lymphocytes CD25 Activated T lymphocyteslmacrophages (Interleukin-2 receptorl CD27 Activated T lymphocytes CD68 Macrophages. monocytes Example 3 Using the method described in Example 1 biopsies were taken from patients with venous leg ulcers and the CD4:CD8 ratios and CD25 levels were determined using the method described in Example 1.
RESULTS
Table 4 shows the CD4:CD8 ratios and CD25 levels detected, together with the observed healing status of the ulcers.
ri,TnT Z, ,, r.r,~l .r~r,ii ,-nr; na anr3 rn7~, 1 PVE!~ s and healincr status Patient CD4:CD8 CD25 Status A 5.7 19.4 Healed B 7.4 17,9 Healed C 6.1 NT Healed D 3.7 t 7.1 Healed E 1.1 10.5 No Improvement 1 F 2.6 13.8 No improvement o G 1.3 11.3 No Improvement H 1.3 12.9 No Improvement I 1,g NT No Improvement i 1,6 5,1 No Improvement I

Example 4 Detection of CD4 and CDS in Chronic Wound Fluid Method Wound fluid was collected from non-healing chronic wounds by absorption into sterile chromatography paper.
Proteins were eluted from the chromatography paper into sterile saline and the saline analysed.

The levels of CD4 and CD8 were determined using Cellf ree (RTM) ELISA kits obtained from T Cell Diagnostics.
The ELISAs were performed in duplicate with a full calibration curve.
S Results Of B samples collected free CD4 could be detected in 6 out of 7 samples and CD8 in 4 out of 8 samples as shown in Table 5 below.
DISCUSSION
These analyses indicate that it is possible to detect the presence of soluble CD4 and CD8 but not CD23 within chronic wound fluid. This is consistent with our immunohistological analysis of non-healing chronic wounds in that CD4' and CD8'T lymphocytes are present in wound tissue but that in the majority of wounds analysed B-lymphocytes, the source of soluble CD23 antigen, are absent.
A wide range of values for CD4 and CD8 were obtained but these were not crrelated to clinical course because they were single time point samples. However the objective of the study was to determine whether detectable levels of lymphocyte derived antigen were present within wound fluid and this has been confirmed.

. - r. . , .. ,.
n n. .. , r : r - , TABLE 5: CD4 and CD8 Detection Sample Protein Activity Activity No. Conc mg/ml ilnits/mg/ml Units/mg/ml 1 1 ..5 55 p 2 21.5 20 75 3 18.5 15 <detection level i 6 1:5 17.5 p AM~MD~D SHEET

Claims (21)

Claims

1. A method of assessing the healing status of a wound in a mammal comprising measuring or detecting the amount of at least one lymphocyte cell membrane antigen present in a sample of body wound fluid or wound tissue.

2. The method of Claim 1 comprising measuring the amount of at least one lymphocyte cell membrane antigen from each of two different types or subsets of T-lymphocytes and assessing the healing status of the wound by comparing the relative proportion of the two types or subsets of T-lymphocytes.

3. The method of Claim 1 or Claim 2, wherein the lymphocyte cell membrane antigen is measured directly using an immunological assay.

4. The method of Claim 3 wherein the assay is selected from the group comprising enzyme linked immunosorbent assay or an immunohistochemical assay.

5. The method of any one of the preceding Claims wherein the cell membrane antigen is an antigen from T-helper lymphocytes.

6. The method of Claim 5 wherein the cell membrane antigen from the T-helper lymphocyte is CD4.

7. The method of any one of Claims 1 to 4 wherein the soluble cell membrane is an antigen from T-suppressor lymphocytes.

8. The method of Claim 7 wherein the cell membrane antigen from the T-suppressor lymphocytes is CD8.

9. The method of any one of Claims 2 and 5 to 8 wherein the healing status of the wound is indicated by the relative proportion of T-helper lymphocytes to T-suppressor lymphocytes.

10. The method of Claim 9 wherein the healing status of the wound is indicated by the ratio of CD4:CD8.

11. The method of Claim 10 wherein a healing wound has a ratio of CD4:CD8 of from 3.0 to 8Ø

12. The method of Claim 10 wherein a non-healing or chronic wound has a ratio of CD4:CD8 of from 0.5. to 2.5.

13. The method of Claim 1 wherein the lymphocyte cell membrane antigen is CD3 or CD25.

14. The method of Claim 1 wherein the cell membrane antigen is an antigen from B lymphocytes.

15. A method for assessing the healing status of a wound in a mammal by monitoring lymphocyte populations within a wound, the method comprising sampling fluid from the wound;
detecting or measuring soluble CD4 T-helper lymphocyte antigen in the sample;
detecting or measuring soluble CD8 T-suppressor lymphocyte antigen in the sample;
wherein the present or absence of healing of the wound is indicated by the relative proportion of CD4:CD8.

16. A method for assessing the healing status of a wound in a mammal by monitoring lymphocyte populations within a wound, the method comprising obtaining a sample of tissue from the wound;
detecting or measuring the proportion of T-helper lymphocytes in the sample;
detecting or measuring the proportion of T-suppressor lymphocytes in the sample;
wherein the presence or absence of healing of the wound is indicated by the relative proportion of T-helper lymphocytes to T-suppressor lymphocytes.

17. A wound healing assessment kit comprising a first antibody that binds to a lymphocyte cell membrane antigen and a labelled antibody that binds to the first antibody.

18. The kit according to Claim 17 which comprises a second antibody that binds to a different lymphocyte cell membrane antigen and a labelled antibody that binds to the second antibody.

19. The kit according Claim 17 or Claim 18 wherein the first antibody binds to the CD4 cell membrane antigen from T-helper lymphocytes.

20. The kit according to Claim 18 wherein the second antibody binds to the CD8 cell membrane antigen from T-suppressor lymphocytes.

21. The kit according to any one claims 17 to 20 which comprises a mean for sampling wound fluid or tissue.