Wealth and antenatal care use: implications for maternal health care utilisation in Ghana

Eric Arthur
Wealth and antenatal care use: implications for maternal health care utilisation in Ghana
R E V I E W
The study investigates the effect of wealth on maternal health care utilization in Ghana via its effect

Introduction and background

The health of women is an important non-income indicator of poverty, hence, one means of reducing poverty in developing countries is to improve the health of women. The Ghana Poverty Reduction Strategy recognizes that improving the health of the poor is crucial for reducing poverty given that, ill health is both a consequence and cause of poverty. According to the Ghana Demographic and Health Survey (GDHS) report [1], "the health care that a mother receives during pregnancy, at the time of delivery, and soon after delivery is important for the survival and well-being of both the mother and her child". Hence, one means of achieving an improvement in the health of women is providing maternal health services, such as antenatal care (ANC) during pregnancy. Indeed the Fifth Millennium Development goal recognizes this and therefore aims to reduce maternal deaths by three-quarters by 2015 (to achieve a maternal mortality ratio of 54 per 100,000 live births) by improving the preventive health care delivered to women during pregnancy. The maternal mortality ratio is high in Ghana. According to the Reproductive and Child Health unit of the Public health division, Ghana health service (RCH/PHD-GHS) [2] the maternal mortality ratio in Ghana is 230/100,000 live births. It is worth mentioning that ANC use alone cannot address the high rate of maternal deaths, but has to be accompanied with a healthy lifestyle by the expectant mother as directed by the health care provider. It is also an indicator of access and use of maternal health care during pregnancy, and serves as a point of contact between the provider and the expectant mother which influences the expectant mother's use of other maternal health services such as delivery and post natal care.

The World Health Organization [3] defines antenatal care (ANC) as "care before birth", and includes education, counseling, screening and treatment to monitor and to promote the well being of the mother and baby. The RCH/PHD-GHS annual report [2], defines ANC as "the health care and education given during pregnancy". The Ministry of Health (MoH) report [4] states that "The objective of antenatal care is to promote and maintain the health of pregnant women. It aims to establish contact with pregnant women in order to detect and manage current health problems. During this period women and their care-givers can develop delivery plans based on their needs, resources and circumstances". It must be stated that, it is only appropriate ANC use that would significantly help in identifying and mitigating the risk factors in pregnancy. Magadi et al [5] posits that the failure to receive appropriate ANC during pregnancy can lead to undesirable pregnancy outcomes such as maternal morbidity, low birth weight for the baby or even maternal and perinatal mortality. The MoH report [4] states that," In order for a particular woman to derive maximum benefit from antenatal care, it is essential for her to start utilizing the service early in pregnancy and to attain a minimum number of contacts with the service". The World Health Organization [6] recommends a minimum of four ANC visits for a pregnant woman before delivery if the woman cannot make the number of visits as directed by the physician/provider. According to Villar et al. [7], recent empirical evidence has shown that four visits suffice for uncomplicated pregnancies, and more visits are only recommended in case of complications. Many mothers in developing countries however do not receive such care, contributing to the high rate of maternal deaths due to pregnancy complications. Antenatal care (ANC) coverage is defined as the percentage of women who used ANC services provided by skilled health personnel for reasons related to pregnancy at least once during pregnancy, as a percentage of live births in a given time period usually one year. The services provided constitutes screening for health and socioeconomic conditions likely to increase the possibility of specific adverse pregnancy outcomes, providing therapeutic interventions known to be effective; and educating pregnant women about planning for safe birth, emergencies during pregnancy and how to deal with them.

ANC services are provided free of charge in all government hospitals in Ghana, (and in some accredited private, NGO and religious hospitals). Some private and religious hospitals also provide ANC services, but at a fee. ANC coverage has been around 87%, but adequate utilization (expectant mothers who are able to attend the minimum of four visits) of the service has been stagnant around 60% for the past four years according to the Ministry of Health report 2007 [4]. The Initiative for Maternal Mortality Programme Assessment (IMMPACT) [8] reported that some of the main causes of death in Ghana are likely to be inaccessible and unaffordable health care. In 1985, as part of the reforms in the financial sector of the economy, user fees were introduced in health facilities in Ghana commonly referred to as the "cash and carry system". This policy led to a reduction in the utilization of services especially among the poor. In September 2003, the policy of exempting users from delivery fees was introduced in the four most deprived regions of the country; Central, Upper East, Upper West and the Northern regions. In April 2005, the policy was extended to the remaining six regions of the country. The aim of the policy was to reduce the financial barriers in using maternity services, (which includes ANC). It was expected that this would lead to a reduction in maternal and perinatal mortality in the country. Maternal mortality is still high even after the introduction of the policy, (IMMPACT) [8]. The policy was introduced with the view that, the cost of maternal services was serving as a hindrance to utilization. It is therefore expected that after the implementation of the free maternal health policy, wealth should have an insignificant effect on the use of ANC services in the country. Indeed Abor & Abekah-Nkrumah [9] and Overbosch et al. [10] who used data prior to the introduction of the free ANC policy found a positive and significant relationship between wealth and ANC use in Ghana. This suggests that probably improving the wealth of individuals or removing the cost barrier would help improve the use of adequate ANC in Ghana. Thus, this study seeks to fill the research gap after the introduction of the policy and to investigate if wealth still has an effect on maternal health services using ANC as a proxy, and hence find out if there are other variables that also influence the use of ANC and hence other maternal health care services in Ghana.

Wealth and antenatal care use

Wealth signifies the economic status of the individual/ family. Several studies have found wealth to influence the use of health services positively. Ortiz [11] concluded that wealthier mothers have more chance of attending a first visit and additional visits than poorer mothers in Colombia. Celik and Hotchkiss [12] showed that household wealth is positively and significantly associated with choosing health facility for delivery. Similarly, Gage [13] found household poverty and personal problems to be negatively related to the use of maternal health care.

Abor & Abekah-Nkrumah [9] found that, as compared to those in the poorest households, those in the poorer households in Ghana are more likely to deliver in a health facility, with those in the middle wealth quintile being more likely to use antenatal services and deliver at a health facility. Wealth is expected to have a positive relationship with ANC since the use of the service is associated with the cost of consultation and the purchase of recommended medication alongside other indirect costs such as transportation cost. Thus, it is expected that, the higher the wealth of the woman, the more likely is she to use ANC because she may be able to afford the cost and other expenses that comes with using the service. The GHDS [1] reports, that there is a "positive relationship between professional antenatal care coverage and wealth quintile, with women in the highest wealth quintile more likely to receive care from a health professional than those in the lowest wealth quintile, although the difference is small (99 and 93 percent, respectively)". The wealth quintile is derived from a wealth index for the household. The index has been divided into five quintiles in the data set; the lowest quintile (poorest), 2nd quintile (poorer), 3rd quintile (middle), 4th quintile (wealthier) and the 5th quintile (wealthiest). The variables that were included in the calculation of the index include: ownership of house-whether the house is owned by the household head or rented, whether there is electricity in the house or not, source of drinking water-pipe borne or well, ownership or a car, the type of cooking fuel usedfirewood or others, location of kitchen-whether indoors or outside and the number of rooms in the house. Hence, with the introduction of the free maternal health policy, it was expected that wealth should have an insignificant effect on maternal health care use. This study therefore seeks to validate this prediction, and further investigate if there are other variables that also influence the use of ANC, and hence maternal health care services in Ghana.

Other socio-economic determinants of antenatal care use

Other socioeconomic variables that do influence the use of ANC are level of education, age, residence (rural/ urban), geographical location (region of residence) and number of living children. These are other potential variables that are likely to either aid the expectant mother in the use of ANC or hinder her. The level of education of the mother is a very key factor that determines the level of utilization of maternal health services. According to Grossman [14], education makes a person efficient in the use of health services and may enable the individual to choose a more health conscious behavior to improve health. Henze [15], Mekonnen & Mekonnen [16], Celik [17], Ortiz [11], Alexandre et al [18], Abor and Abekah-Nkrumah [9], Tayie and Lartey [19] and Addai [20] are some studies that have found a positive and significant association between education and maternal health care use. Overbosch et al [10], concluded in Ghana that, "Women's attitude to antenatal care seems to be influenced by their schooling, since more years of education of a pregnant woman is associated with a choice for sufficient antenatal care". Thus, in the campaign to raise the utilization of maternal health care services, there is the need to encourage women to pursue higher education.

Age and the number of living children of the mother may also affect her use of ANC. Empirical studies on age presents mixed evidence. For instance Chandhiok et al [21] and Henze [15], found a reduction in the proportion of women obtaining ANC services with increasing age in India and Honduras respectively; on the other hand Celik [12] and Ortiz [11] found a positive relationship between ANC and age in Turkey and Colombia respectively. Thus, the influence of age on the use of maternal health cannot be determined prior to investigation. Alongside age is the number of living children of the expectant mother which proxies a mother's experience with ANC services. The expectant mothers' use of ANC may be influenced by her previous experience. This may be positive ( a pleasant experience at the health center) increasing the use of the service or negative ( an unpleasant experience at the health center), thereby reducing her use of ANC. Complications experienced during earlier pregnancies also have a positive association with the use of ANC. According to Overbosch et al [10], "Pregnancy is a natural process and women with some experience might consider antenatal care less necessary". This may be due to the fact that after encountering the service, the expectant mother may think that she knows enough to take care of subsequent pregnancies. Elo [22] and Raghupathy [23] report that a higher number of previous pregnancies are associated with less use of ANC, while Magadi, Madise and Rodrigues [5] report a negative association between a higher number of previous pregnancies and early attendance to antenatal care.

Place of residence (rural/urban) and Geographical location (region) may also affect the utilization of ANC services. According to Abor & Abekah-Nkrumah [9], urban dwellers may be relatively closer to healthcare facilities than rural dwellers in most developing countries. Overbosch et. al., [10], reports that "currently, more than a third of the rural women have to travel more than 5km to the modern provider of ANC" in Ghana. Thus, accessibility to health care services may be much easier for the urban dwellers than the rural dwellers, thereby increasing the probability of an expectant mother in the urban center using ANC compared to her rural counterpart. Celik & Hotchkiss [12], Navaneetham & Dharmalingam [24] and Abor & Abekah-Nkrumah [9] are some studies that have concluded that differential access to health care facilities between the rural and urban centers reduced utilization of maternal health care services for the rural dwellers. Closely related to this is the distance to the health facility and transportation problems one may encounter in accessing health services. Thus, these can greatly hinder the utilization of the ANC services since they serve to discourage the expectant mother who may have to travel along bad road networks or may have to travel for long distance before being able to access a health centre for ANC. Even if the mother does it the first time, further visits may be hindered due to the struggle to get to the health facility, thereby reducing adequate utilization.

Data and methods

Data

The study uses data from the 2008 Ghana Demographic and Health Survey (GDHS 2008). The survey is carried out every five years (In Ghana, it was carried out in 1988,1993,1998,2003,2008). A special survey on maternal health was carried out in 2007. The sample contains women within the ages of 15-49 with a live birth in the five years preceding the survey. The questionnaire consists of questions on demographic indicators, health status, illnesses, and visits to a doctor, health behavior such as questions on smoking, drinking alcohol, physical activity, and eating habits. To measure demand for ANC the number of visits to a modern health facility namely public/government hospitals, mission hospitals and private hospitals is used. This has however been ordered according to the WHO [6] protocol of a minimum of four visits before delivery, which is seen as sufficient to ensure safe delivery, and further visits are recommended only when any complications are detected by the physician. The price of ANC is not included in the estimation equations, because ANC services are provided free of charge in all public hospitals, and some private and religious hospitals for all pregnant women in Ghana. Hence an expectant mother who decides to use a private hospital that charges for the service is revealing her preference for the services of that health provider and this may be due to other factors not covered in this study. In this study, a total of 2118 women who had given birth prior to the five years preceding the survey are investigated.

Methods

The study employs both univariate and multivariate analysis to investigate the effect of wealth and other socioeconomic variables on ANC use in Ghana. The univariate analysis involves the use of chi square to test the significance of association between ANC use and each of the predictors in the study. In reality there are a multiplicity of factors that do influence the mother in making such a decision, thus, the study goes a step further to carry out a multivariate analysis when these variables are allowed to interplay. The multivariate model is specified as a generic latent linear probability function as:
H* ¼ X'β þ uð1Þ
H* is the number of antenatal care visits (which has been ordered as 0, 1, 2, 3, and 4 plus visits); X is a vector of explanatory variables, and U represents the random error term that is assumed to be normally distributed.

The model above is estimated using the ordered logistic regression model. This is an extension of the logistic regression model, allowing for more than two responses. It is estimated using maximum likelihood estimation technique. The ordered logistic model is employed in this study because the dependent variable, ANC visits has been put in an ordered discrete choice from no visit to four or more visits as proposed by the W.H.O. This is to help find out the influence of wealth and other socio economic variables which influences the woman's decision as she progresses from no visit to the recommended number of four visits. The control variables are education, number of living children, age of the mother, and distance to health facility, problems of transportation, residence and regional dummies.

Results

The Pearson's chi square test is used to test whether there is a significant difference in the use ANC by the various explanatory variables. The results as presented in Table 1 indicate that there is a significant relationship between each of the explanatory variables and ANC use except age and employment status. The use of ANC improves with the wealth and educational level of the woman. Utilization is also higher among women in the urban areas and expectant mothers with health insurance. The results also suggest that ANC use falls with an increasing number of living children. The table also shows that Greater Accra region recorded the maximum number of expectant mothers who made the recommended visits compared to the Ashanti and the Upper West Regions with 85.4% and 85.2% respectively. The Northern Region however recorded the lowest number of the mothers who made the recommended visits (67.5%). It must however be noted that this analysis was carried out for each of the explanatory variables without controlling for the other factors that also influence the use of ANC. In the next sub-section, a multivariate analysis is conducted to assess the impact of each explanatory variable on ANC use whilst controlling for the other factors that also influence ANC use in Ghana.

The results in Table 2 suggest that wealth, education, number of living children, problems with transportation, ownership of health insurance, age of woman and residence (rural/urban) are important predictors of the use of ANC in Ghana. The results also show regional variations in the use of ANC and differential use between the rural and urban dwellers. Thus, these variables together do influence the use of ANC and other maternal health services in Ghana, and therefore any targeted intervention should focus on these characteristics to be able to achieve the desired results of reducing maternal mortality through child birth.

Discussion of results

The paper sought to investigate the impact of wealth and other socio economic determinants of maternal health care use via ANC use in Ghana since the inception of the free maternal health policy to reduce financial barriers in the use of maternal health services. The introduction of the free maternal health policy was with the assumption that there was a significant financial barrier to the use of maternal health services, and this contributed to the high incidence of maternal mortality in the country. Hence, offering a free service will help expectant mothers in their use of maternal health services. In such a case, the effect of wealth on the use of the service should be insignificant. The results however suggest that wealth still has a positive and significant influence on the use of ANC, contrary to expectation that it should not since the service is free. Women in higher wealth quintiles are more likely to make more ANC visits than women in the lowest wealth quintile. We can infer that even though the service is provided freely, it may come with costs either directly or indirectly and those with the resources are more likely to afford it. In general, it can be concluded that, even though maternal health care services are rendered free of charge in Ghana, wealth which signifies the financial position of the individual is still a challenge in the use of these services. It still hinders the utilization rates; expectant mothers may still use the services, but not adequately as recommended by the WHO (in the case of ANC minimum of four visits before delivery) to ward off any health effects of child birth and hence to reduce the rate of maternal and child deaths through delivery. Thus, to improve the use of ANC, there is the need to go beyond providing the free services to finding means of support for expectant mothers. This can be in the form of the provision of necessary drugs and more importantly ensuring that these recommended drugs are available at the health facility to the expectant mothers. This will help reduce the cost involved in purchasing the drugs when it is not available because this can hinder the mother's use of the services. Again, most of the expectant mothers end up spending the entire day at the health center for their check-ups. This is a form of indirect cost especially to the mothers in the informal sector who may have to go to the market to earn a daily living. Hence, there may be the need to increase the number of physicians/service providers attending to them at the health center so that they would be able to leave early. Thus, these can be done to complement the free delivery policy, and can help improve the use of adequate ANC and other maternal health services, and therefore reduce the rate of maternal deaths through child birth in Ghana. Education, residence and transportation to the health centre have also been found to influence the use of ANC. Women with secondary or higher levels of education are more likely to use adequate ANC compared to those without education. Thus, improving the education of the mother in Ghana, will contribute greatly to the use of maternal and ANC services by women and thus help in reducing maternal and child mortality in Ghana. Specifically, women should be encouraged to pursue education beyond the primary level as the study has found that women with higher levels of education tend to make adequate use of ANC. Also, it may be possible that the less educated (especially in the rural centers) are not adequately informed about the services being offered freely and also about the adequate utilization for each pregnancy, hence it may be necessary for the National Commission for Civic Education in Ghana (NCCE) to intensify the education on the use of maternal health care services probably through the mass media (radio, television, print media) and the community announcements, especially in the rural centers where the use of mass media may not be very effective. Thus, we need to continue educating expectant mothers and women in general on a daily basis on the use of these maternal health services and to increase awareness in the communities. Expectant mothers in urban areas also tend to use more ANC compared to their counterparts in the rural areas. The positive effect might indicate that distribution of facilities between the urban and the rural areas are in favor of the urban dwellers, putting the rural dwellers at a disadvantage in terms of availability and accessibility of the services provided. Hence the Government, NGO's and religious health care providers should make an effort to set up health centers in the rural areas to help improve utilization. This may help to also eliminate the problem of transportation if these facilities are set up in close range for the use of the rural communities. This also applies to the various regions as there exist considerable regional variations in the use of ANC.

The age of the expectant mother, number of living children and health insurance are other factors that also influence the use of ANC in Ghana. Older women are more likely to use more ANC compared to younger women. Following the predictions of the Grossman model [22]), age increases the rate of depreciation of the health of the individual, it may therefore be possible that, the older women may patronize health services more than the younger ones, and ANC services will not be an exception. There is therefore the need to educate the younger mothers on the need to use ANC and other maternal health services. ANC use also falls with increasing number of children. This may be due to the fact that once a woman has gone through the experience of child birth, she may be reluctant to undertake ANC visits for later pregnancies either due to a bad experience with the service or probably she begins to think that she has an idea of what is required of her during pregnancy. Hence, the campaign to increase the use of ANC should place a lot of focus on expectant mothers who have had the experience of child birth on the need to use adequate ANC for each pregnancy to avoid any health effects of childbirth. Expectant mothers with health insurance tend to use more ANC than those without health insurance.

Conclusion

The study investigated the effect of wealth on maternal health care use via the use of Antenatal care (ANC) in Ghana using data from the 2008 Ghana Demographic and Health Survey (GDHS 2008). The study hypothesized that, wealth does not influence utilization of ANC services since the introduction of the free maternal health care policy which sought to remove the cost barriers to the use of ANC. Hence, expectant mothers are expected to achieve the minimum recommended number of four visits per the World Health Organization (W.H.O) protocol before delivery. Using both univariate and multivariate analysis the results have suggested that wealth significantly influences the use of ANC in Ghana. Thus, even though the free maternal health policy may have increased the utilization of ANC and maternal health care services in Ghana, adequate utilization has still not been achieved because affordability as proxied by wealth is still a problem to some expectant mothers, especially, those within the lowest wealth quintiles. It is thus recommended that to ensure adequate use of ANC and maternal health care in Ghana, some means of support is provided to women within the lowest wealth quintiles alongside the policy. This can be the provision of necessary medication prescribed for the expectant mothers, and ensuring that such drugs are at least available at the health center to relieve the mother of the cost she may have to incur in purchasing the medicine, and the availability of a number of Physicians to ensure that the mother does not spend too much time since this will serve as a discouragement to the mother in the use of the service, especially those in the informal sector, who may have to go to the market and sell to support the family as this is the case in most rural communities in Ghana. Policy should also focus on encouraging women to pursue education to at least the secondary level and the National Commission for Civic Education (NCCE) should endeavor to intensify the campaign on the use of maternal health care, especially on the free provision of the service. This may be done through the mass media (Television, radio, etc) and community announcement especially in the rural communities, since such communities are not likely to have adequate access to the mass media. There is the need to intensify education especially for the mothers who have had the experience of child birth on the need to use adequate ANC for each pregnancy. Such mothers from the study tend to use less ANC and maternal health services in general. These measures can be done alongside the policy, and hence would help in reducing the rate of maternal deaths due to child birth, and help Ghana achieve the Millennium Development Goal (Goal 5) of reducing maternal mortality.

Competing interest

The author declares no competing interest.

Author's contribution

The author conceived the study, undertook the analysis and did the write up of the manuscript. The author read and approved the final manuscript.

Table 1
1Univariate investigation of antenatal care use in Ghana *** indicates there exists significant differences within that predictor in the use of ANC.Variables
No ANC (%)
1-3 visits (%)
At least 4 visits (%)
Chi-square test

Educational level of the mother
82.7***

No Education
5.5
22.8
71.7

Primary Education
5,0
20.0
75.0

Secondary Education
1.9
10.2
88.0

Higher Education
0.0
0.0
100

Wealth Quintiles
145.8***

Lowest
6.2
25.8
68.0

2nd
5.6
20.4
74.0

3rd
3.0
17.7
79.3

4th
1.3
6.5
92.2

Wealthiest
0.4
3.3
96.3

Residence

Urban
1.3
8.2
90.5
89.8***

Rural
5.3
21.6
73.1

Health Insurance

No
5.3
21.5
73.2
68.2***

Yes
1.8
10.4
87.8

Employment status

Not Employed
1.5
17.2
81.3
4.61

Employed
4.2
16.8
79

Region

Western Region
5.4
20.3
74.3
85.1***

Central Region
3.9
19.2
76.9

Greater Accra Region
2.4
4.4
93.1

Eastern Region
3.3
22.8
73.9

Ashanti Region
1.9
12.7
85.4

Brong Ahafo Region
2.9
16.9
80.2

Northern Region
4.9
27.5
67.5

Upper East Region
4
13.6
82.5

Upper West Region
2.9
11.9
85.2

Age of woman

19 years and below
1.9
24.5
73.6
6.06

Between 20 and 35 years
3.8
16.6
79.7

35 years and above
4.4
16.1
79.5

Number of Children

1 child
1.5
15.0
83.5
27.2 ***

2 -4 children
3.6
15.3
81.0

5 or more children
6.1
21.0
72.9

Table 2
2Multivariate investigation of antenatal care use in GhanaVariable
Coefficients
Linearised Standard Errors
t stat.
P value

Wealth Quintiles (Ref: Lowest quintile)

2nd quintile
0.24
0.14
1.79*
0.07

3rd quintile
0.42
0.16
2.66**
0.01

4th quintile
0.90
0.17
5.38***
0.00

Wealthiest quintile
1.32
0.19
6.79***
0.00

Educational level of the mother (Ref: No education)

Primary
-0.07
0.13
-0.50
0.62

Secondary plus
0.24
0.14
1.69*
0.09

Other socio economic indicators

Distance is a problem to health facility
0.10
0.15
0.70
0.49

Transport is a problem to health facility
-0.31
0.15
-2.05**
0.04

Number of living children
-0.15
0.03
-4.37***
0.00

Residence (ref: Rural)
0.28
0.14
2.05**
0.04

Age of Woman in years
0.05
0.01
4.68***
0.00

Health Insurance (Ref: Insured)
0.40
0.09
4.48***
0.00

Employment Status (Ref: Not employed)
0.15
0.14
1.09
0.27

Administrative Regions (Ref: Ashanti region)

Western Region
0.23
0.24
0.96
0.34

Central Region
-0.08
0.24
-0.36
0.72

Greater Accra Region
0.32
0.20
1.60
0.11

Volta Region
-0.24
0.22
-1.09
0.28

Eastern Region
-0.40
0.20
-1.95**
0.05

Brong Ahafo Region
0.18
0.21
0.82
0.41

Northern Region
-0.24
0.18
-1.30
0.19

Upper East Region
0.79
0.31
2.50**
0.01

Upper West Region
0.44
0.20
2.20**
0.03

Number of Observation = 2118 F( 22, 386) = 17.82 Prob > F = 0.000.
***indicates significant at 1% ** significant at 5%, * significant at 10%.

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Cite this article as: Arthur: Wealth and antenatal care use: implications for maternal health care utilisation in Ghana. Health Economics Review. 214Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.comCite this article as: Arthur: Wealth and antenatal care use: implications for maternal health care utilisation in Ghana. Health Economics Review 2012 2:14. Submit your manuscript to a journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com


Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study

Jakob Albrethsen
Department of Clinical Biochemistry, Glostrup Hospital
2600GlostrupDenmark

Rikke Bøgebo rikkebøgeboemail@example.com
Department of Clinical Biochemistry, Glostrup Hospital
2600GlostrupDenmark

Steen Gammeltoft
Department of Clinical Biochemistry, Glostrup Hospital
2600GlostrupDenmark

Jesper Olsen
Surgical department D
Glostrup Hospital
2600GlostrupDenmark

Benny Winther email@example.com
Department of Clinical Biochemistry, Glostrup Hospital
2600GlostrupDenmark

Hans Raskov email@example.com
Colotech ltd
Fruebjergvej 32100Copenhagen Science Park Symbion, CopenhagenDenmark

BMC Cancer

Upregulated expression of human neutrophil peptides 1, 2 and 3 (HNP 1-3) in colon cancer serum and tumours: a biomarker study
10.1186/1471-2407-5-8BioMed Central Page 1 of 10 (page number not for citation purposes) Research article * Corresponding author
Background: Molecular markers for localized colon tumours and for prognosis following therapy are needed. Proteomics research is currently producing numerous biomarker studies with clinical potential. We investigate the protein composition of plasma and of tumour extracts with the aim of identifying biomarkers for colon cancer.Methods: By Surface Enhanced Laser Desorption/Ionisation -Time Of Flight / Mass spectrometry (SELDI-TOF/MS) we compare the protein profiles of colon cancer serum with serum from healthy individuals and the protein profiles of colon tumours with normal colon tissue. By size exclusion chromatography, we investigate the binding of HNP 1-3 to high mass plasma proteins. By microflow we investigate the effect of HNP 1-3 on mammalian cells.Results:Human Neutrophil Peptides -1, -2 and -3 (HNP 1-3), also known as alfa-defensin-1, -2 and -3, are present in elevated concentrations in serum from colon cancer patients and in protein extracts from colon tumours. A fraction of HNP 1-3 in serum is bound to unidentified high mass plasma proteins. HNP 1-3 purified from colon tumours are lethal to mammalian cells.Conclusions: HNP 1-3 may serve as blood markers for colon cancer in combination with other diagnostic tools. We propose that HNP 1-3 are carried into the bloodstream by attaching to high mass plasma proteins in the tumour microenvironment. We discuss the effect of HNP 1-3 on tumour progression.

Background

The diagnostic stage of colon cancer determines survival. Patients diagnosed with localized tumours have a 75% probability of 5 year survival, whereas patients diagnosed with distant metastases only have a 5-10 % probability of 5 year survival (reviewed in [1]). Recently a number of studies have been published in which Surface Enhanced Laser Desorption/Ionisation-Time Of Flight/Mass Spectrometry (SELDI-TOF/MS) has been applied to biological samples from patients with various forms of cancer [2][3][4] leading to the identification of protein markers with clinical potential. Here we present a SELDI-TOF/MS study of colon cancer serum and tumours. We show that the expression of Human Neutrophil Peptides -1, -2 and -3 (HNP 1 -3), also known as alfa-defensin-1, -2 and -3, is upregulated in the tumour microenvironment, as compared to normal colon tissue. This finding is reflected in serum. We find that HNP 1-3 is present in elevated concentrations in serum from patients diagnosed with tumours in the colon, as compared to serum from a healthy control group matched by age and gender. By sizeexclusion analysis we add to the existing evidence that HNP 1-3 bind to high mass plasma proteins, explaining the presence of HNP 1-3 in serum. By microflow analysis, we show that HNP 1-3 purified from colon tumours are lethal to mammalian cells. The HNP 1-3 peptides are part of the defensin family of peptides (reviewed in [5][6][7]), which are a fundamental component of the immune system and have the capacity to kill / inactivate a broad range of pathogens. Defensins are also known to function as regulators of both the innate and the adaptive immune system. We discuss the possible effects of HNP 1-3 in the tumour microenvironment.

Methods

Biological samples

All biological samples were obtained by trained staff at Glostrup Hospital, Denmark. Written consent was obtained from all donators. Permission was obtained from the Danish Scientific Ethical Committee and the Danish Data Protection Agency.

Tissue screening

Normal colon tissue samples and colon tumour samples were obtained from the removed fragment of the patient's colon after surgical treatment for colon cancer. Tissue samples were stored at -80°C until use. The protein content was extracted from the tissue: 100 mg tissue sample was thawed on ice and homogenised on a Wheaton Overhead Stirrer for 2 minutes at speed step 2, in 500 ul Lysis buffer (100 mM TRIS-HCl, pH 8.0, 9.5 M UREA, 2% CHAPS). The samples were centrifuged at 14,000 rpm for 10 minutes and the pellet was discarded (repeated twice). The tissue protein extracts were stored at -80°C until use. Pilot studies were performed on different chips (data not shown) and the NP20 (Normal Phase) (Ciphergen) chip was chosen for the tissue screening. NP20 chips were placed in Bioprocessor (Ciphergen) and pre-treated with 50 ul tissue binding buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes on shaker (250 rpm) (repeated twice). 5 ul tissue protein extract was diluted in 50 ul tissue binding buffer and incubated in Bioprocessor on NP20 chips for 40 minutes at room temperature on shaker (250 rpm). Spots were washed twice in 250 ul tissue washing buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes. The chips were air dried for 10 minutes, followed by treatment with two times 0.6 ul 100% sinapinic (SPA) matrix solution. SPA was obtained from Ciphergen in 5 mg aliquots and dissolved (150 ul MQ water, 150 ul acetonitrile, 1.5 ul Tri-Fluoro-acetic-Acid (TFA)) immediately before the screenings.

Serum screening

Colon cancer serum samples were obtained from patients before surgical treatment. Normal serum was obtained from a group of healthy individuals matched by age and gender to the cancer patients. All serum samples were stored at -80°C until use. Serum pilot studies were performed on different chips to monitor the presence of HNP 1-3 in serum (data not shown). The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice). The chips were placed in Bioprocessor and incubated with 100 ul MQ for 5 minutes on shaker (250 rpm). Each spot was treated with 50 ul serum binding buffer (100 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 0,1% Triton X-100) and left on shaker for 5 minutes (250 rpm). Serum samples were thawed on ice and 1 ul serum was diluted in 50 ul serum binding buffer and applied to spots and left on shaker (250 rpm) at room temperature for 40 minutes. The sample solution was removed and the spots were washed twice in 200 ul serum washing buffer (100 mM PBS, pH 7.4, 700 mM NaCl), followed by one wash in 200 ul MQ water. The chips were removed from the Bioprocessor and left to air dry for 20 minutes followed by treatment with two times 0.6 ul SPA.

Only freshly made matrix solutions were used and the instrument was calibrated daily. Cancer and normal samples were run side by side. The chips were analysed on a PBS II instrument (Ciphergen). All spectra in each screening were normalised based on total ion current.

Purification and identification of HNP 1-3 100 ul protein extract from colon tumour tissue in tissue lysis buffer was loaded unto a RP-HPLC column (uRPC C2/C18 ST 4.6/100, Pharmacia Biotech, Flow rate: 0.5 ml/ min, Fraction size: 0.5 ml) in buffer A (0.065% TFA in MQ-water) and proteins were eluted in a gradient of 0-100% buffer B (0.05% TFA in acetonitrile (ACN)). Elution of peptides was monitored by absorbance at 280 nm. All protein-containing fractions were analysed by Matrix Assisted Laser Desorption/Ionisation-Time of flight (MALDI-TOF) on the PBS II instrument: 1.5 ul fraction was incubated with 0.6 ul SPA on a Gold array (Ciphergen) and left to crystallise, followed by an additional treatment with 0.6 ul SPA and analysed in the PBSII instrument. The HNP 1-3 containing fraction (32% buffer B) was further purified on a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, Flow rate 0.9 ml/min, Fraction size: 0.5 ml, Buffer: 50% ACN, 0.1 % TFA). Elution of peptides was monitored by absorbance at 280 nm and protein-containing fractions were again analysed by MALDI-TOF. Purified HNP 1-3 were identified by on-chip trypsin digestion: 10 ul of HNP 1-3 fraction was applied to an NP20 chip and left on shaker (250 rpm) at room temperature for 40 minutes. The solution was removed and the spot was washed twice with 10 ul water. In order to denature peptides prior to digestion, the chip was left on heating block (80°C) for 5 minutes. The chip was cooled on ice for 2 minutes. 10 ul trypsin digestion solution (0.01 ug/ul trypsin in 50 mM NH 4 HCO 3 , pH 8,0) was added, and the chip was left for 10 hours at 40°C in humidity chamber after which the chip was left to air dry for 20 minutes. 1 ul CHCA matrix (prepared as the SPA matrix solution) was added and the peptide map was analysed on the PBS II instrument. Peptide maps of trypsin autodiggest were used as controls. Identification was done with the PepIdent software on the Expasy server. For the reduction experiment, HNP 1-3 were first denaturation by heating (10 minutes at 80°C) followed by treatment with DTT (200 mM, 30 minutes at room temperature) and the peptides were incubated on an NP20 chip and analysed on the PBS II instrument.

Size exclusion chromatography of HNP 1-3

50 ul colon cancer serum was loaded unto a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, optimal separation range: 1 to 7 kDa, flow rate: 0.5 ml/min, fraction size: 0.5 ml, buffer: 10 mM Ammonium carbonate, pH: 8.0). Elution of peptides was followed by absorbance at 280 nm. All protein containing fractions were analysed by MALDI-TOF on PBS II (Ciphergen) as described above. Maximum signal intensity of 40 individual peaks was plotted as a function of elution volume and an approximate elution curve was calculated.

Study of HNP 1-3 by microflow

For micro flow experiments, canine kidney cells (MDCK cells) were plated onto poly-d-lysine coated cover slips at a concentration 3000 cells/well, grown in Dilbeccoo's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) for five days with the result of confluent islands. Microflow was performed in an Eppendorf micromanipulator 5171 and transjector 5246 system mounted on a Leica DMIRBE inverted research microscope. Micro capillaries (borosilicate with filament, Sutter Instruments Company, Novato, California, USA) were pulled to an outer diameter of 0.85 nm on a Sutter P-97 Micropipette Puller. The dye-loaded cells were visualized by excitation at 470 nm and recorded at 509-nm emission using Haupage version 3.3.18038 software and Kappa CF 15/4 MC-S camera (Leica). The MDCK cells were recorded (in CO2 independent media) on the inverted DMIRBE inverted research microscope. The capillary was placed 20 nmover the confluent cells with a constant flow (1300 hPa). The MDCK cells were exposed to peptide and calcein (20 mM) fractions for 60 minutes.

Results

HNP 1-3 expression in tissue and serum

We performed pilot studies of colon tumour and normal colon tissue on a variety of chips with different chemical properties and with different binding and washing conditions. Based on these preliminary studies, we found that the expression of three peptides with mass/charge ratio (m/z) values of 3372, 3443 and 3486 (subsequently identified as HNP 2, 1 and 3, respectively), were upregulated in the tumour samples. The three peptides were visible on different chips and under different binding conditions (data not shown). The strongest signal of HNP 1-3 in tissue extract was obtained on the NP20 (Normal Phase) chip, whereas the strongest signal of HNP 1-3 in serum was observed on the IMAC30 (Ni) (immobilised metal affinity capture chip, activated with nickel), and these conditions were chosen for the actual screenings. We emphasize that in general the protein profiles of serum and tissue were very different when using the same protein chip for serum and tissue extract. However, individual peaks were present on several types of chips, and observed in both serum and in tissue extract, for example the characteristic triplet with m/z 3372, 3443 and 3486. Protein extract from 40 colon tumour and 40 normal colon tissue samples were analysed on NP20 chips and 125 colon cancer serum samples and 100 normal serum samples were analysed on IMAC30 (Ni) chips. All spectres in each screening were pooled and normalised based on overall ion current. Each spectrum produced approximately 40 to 90 protein peaks in the range from 2 to 80 kDa (FIG. 1A-C). Statistical analysis of the intensity values of HNP 1-3 in the tissue screening ( FIG. 2A.) showed that HNP 1-3 were significantly upregulated in tumours (p < 0.0005) and statistical analysis of HNP 1-3 expression in the serum screening (FIG. 2B.) showed that HNP 1-3 were significantly upregulated in cancer serum (p < 2.2e -16 ).

Compared to other peptides in the same range, HNP 1-3 showed average signal intensity in most normal colon tissue extract, whereas the HNP 1-3 signal was extremely high in most tumour samples (in some tumour spectres the HNP 1-3 peaks were the strongest of all peaks). In the normal serum samples the HNP 1-3 signals were weak and only slightly stronger in the cancer serum.

Identification of HNP 1-3

The markers were purified by RP-HPLC, peptide gelfiltration and on-chip purification, after which they were identified by peptide mapping as HNP-2 (3372 Da), HNP-1 (3442 Da) and HNP-3 (3486 Da) ( Table 1A). The masses correspond to the peptides in their oxidised states with three disulfide bridges. After reduction with DTT, HNP-1 and HNP-2 increased 6 Daltons in mass, due to reduction of the six cysteines (Table 1B). We were not able to reduce HNP-3.

Size exclusion chromatography of HNP 1-3

50 ul colon tumour extract in Lysis buffer was applied to a peptide gelfiltration column. Elution of peptides was followed by absorbance at 280 nm. All fractions were analysed by MALDI-TOF on the PBS II instrument (Ciphergen). Maximum signal intensity of 40 individual peaks was plotted as a function of the elution volume and an approximate elution curve was calculated (FIG. 3). HNP 1-3 peptides were primarily eluted in early fraction together with high mass proteins above 20 kDa and also, but to a lesser degree, in fractions together with other peptides of similar mass interval (2 to 4 kDa) (FIG. 3).

Cytoxic assay

The cytotoxicity of HNP 1-3 purified from colon tumours was tested by exposing MDCK cells to different fractions purified from colon tumours. Calcein were added to the fractions and the solutions were left to flow over the cells for one hour. By fluorescence microscopy calcein was observed to accumulate in cells exposed to HNP 1-3/calcein fractions, whereas cells treated with fractions containing other (unidentified) tissue peptides did not uptake calcein (FIG. 4C&D). Further, by microscopy we observed that cells exposed to HNP 1-3 appeared more diffuse and had enlarged nuclei, indicating apoptosis (FIG. 4A&B).

Discussion

Elevated concentrations of HNP 1-3 in colon cancer serum

Abnormal concentration of HNP 1-3 in blood has previously been demonstrated in connection with benign conditions. Elevated concentrations of HNP 1-3 following infection (bacterial-/ non-bacterial-infection and pulmonary tuberculosis) has been found in plasma, blood and other body fluids [8], and plasma HNP 1-3 concentrations have been shown to be elevated in patients with septicemia or bacterial meningitis [9]. HNP 1-3 have been found in urine from patients with transitional cell carcinoma of the bladder [10] and HNP-1 has been found in salvia of patients with oral carcinomas [11]. Finally, HNP 1-3 are found in excess amounts in tears after ocular Protein profiles of serum and tissue Figure 1 Protein profiles of serum and tissue. Representative SELDI-TOF/MS spectra of normal colon tissue (FIG. 1A) on the NP20 chip and normal serum (FIG. 1B) on the IMAC30 (Ni) chip. The two spectra differ significantly and each produce a total of 40 to 60 peaks. The majority of the peaks are present in the specified range from 2 to 10 kDa. FIG. 1C Comparison of a typical colon tumor spectrum (above) and a normal colon spectrum (below) in the range from 3 to 4 kDa. The arrows point to the three differentially expressed peptides, subsequently identified as HNP 1-3. The three peptides are expressed in normal colon tissue and the expression is upregulated in the tumor samples. The three peptides are present in normal serum, but are present in higher concentrations in colon cancer serum. The average peak intensity for the three peptides were significantly lower in serum than in tissue. Figure 2 Expression of HNP 1-3 in tumors and serum. FIG.2A HNP 1-3 profiles of normal and colon tumor tissue. 40 colon tumor and 40 normal colon tissue samples were analysed on NP20 chips. Differences in mean intensities of HNP 1-3 in normal and colon tumor tissue were statistical significant at 5% level (p < 0.0005). FIG. 2B HNP profiles of normal and colon cancer serum. Serum samples (125 colon cancer and 100 normal) were analyzed on IMAC30 chips. The mean intensities were significantly different at 5% level (p < 2.2e-16). The reproducibility was significant for all three peptides in both tissue and serum. The standard deviation of HNP 1, 2 and 3 was 70%, 136% and 57% in normal tissue and 11%, 15% and 8% in tumor tissue. The standard deviation of HNP 1, 2 and 3 was 96%, 154% and 81% in normal serum and 234%, 365% and 282% in cancer serum. The boxplot show the 25th quantile, median, 75th quantile, and whiskers extent to min. and max. values.

Expression of HNP 1-3 in tumors and serum

surface surgery [12]. Our study is the first that demonstrate elevated concentrations of HNP 1-3 in blood following tumour growth.

Elevated concentrations of HNP 1-3 in colon tumours

HNP expression has previously been linked to different types of tumours and cell lines. HNP-1 has been detected in lung tumours [13] and in the submandibular glands of patients with oral carcinomas [14]. By RT-PCR, mass spectrometry and flow cytometric analysis, HNP 1-3 have been shown to be expressed by cell lines deriving from renal cell carcinomas [15] and the expression of a specific HNP precursor peptide has been shown to be upregulated in human leukemic cells [16]. Our results suggest that HNP 1-3 are extremely abundant in colon tumours. This is in agreement with a study of HNP-1 in lung tumours, where the maximum observed level was 26 nanomoles per gram wet tissue [13]. In order for these excessive amounts of peptide to be detectable in serum, the peptides must be present extracellular in the tumour environment, such as observed in studies of HNP 1-3 expression in kidney [14] and brain [17]. Usually tumour expressed peptides are not easily detected in serum or plasma by SELDI-TOF mass spectrometry techniques; the highly concentrated plasma proteins out compete the signal of low abundance peptides. We suggest that HNP 1-3 are detectable in serum only because they are expressed in exceptionally high amounts in the tumour microenvironment.

Previous studies indicate that HNP 1-3 expression in tumours primarily originate from tumour invading eosinophils [13] and neutrophils [14,18]. However, it has been shown that the excess amounts of HNP 1-3 observed in urine from bladder cancer patients were often produced by the actual bladder cancer cells [10], and that highly invasive bladder cancer cells produced more HNP 1-3 than less invasive ones (Holterman DA, in press, personal communication). Since our tissue screening is based on comparison of tissue samples, we cannot say whether the peptides are produced by the colon cancer cells or by tumour infiltrating neutrophils. In the case of active inflammatory bowel disease, it is not clear whether epithelial expression of HNP1-3 is induced by the inflammatory state or the whether the peptides are released by adjacent neutrophils and taken up by the epithelial cells [6]. Here it is believed that the peptides provide protection against microbial invasion when the mucosal barrier is damaged (such as is the case in inflammatory bowel diseases). HNP 1-3 are known to stimulate bronchial epithelial cells to upregulate interleukin-8 production [19], a potent neutrophil chemotactic factor. Thus, the upregulated expression of HNP 1-3 in tumours may primarily originate from invading immune cells, but could be initiated by HNP 1-3 producing cancer cells.

Size exclusion chromatography of HNP 1-3

We explain the elevated concentrations of HNP 1-3 in colon cancer serum by unspecific binding between HNP 1-3 and high mass plasma proteins. We suggest that the peptides attach to plasma proteins in the tumour area and are carried into the bloodstream. The HNP 1-3 we observe in high mass fractions from size exclusion, could also be explained by multimerisation: In one study it was demonstrated that defensins form voltage dependent channels in Table 1: Identification of HNP 1-3. Table 1A HNP 1-3 peptides were identified by peptide mapping (on-chip trypsin digest). Three sequence specific fragments were produced (615.8, 930.6, 1061.4 Da.). The peptides were identified as HNP 1-3 using the PepIdent server on the ExPASy homepage. Table 1B The measured masses of the HNP 1-3 peptides were approximately 6 Daltons lower than the expected theoretical masses. This deviation was caused by the in vivo formation of three disulfide bridges. By heat denaturation and treatment with DTT we were able to protonate the six cysteines in HNP 1 and 2. (HNP-3 was consistently degraded during the reduction procedure). lipid bilayer membranes and further conductance investigations suggested that the channels were formed by multimers containing 2-4 molecules [20] and a crystal structure study [21] of HNP-3 revealed an amphiphilic dimmer. We interpret the size exclusion results as evidence for interaction between HNP 1-3 and unidentified high mass proteins through unspecific interactions; the peptides are eluted in very early fractions which would probably require the presence of multimers of more than five peptides and our study does not reveal the presence of multimers containing four, three or two peptides. Further, our interpretation is in agreement with a number of previous studies that show that HNPs are bound to plasma protein in vitro and that high concentrations of HNPs causes precipitation of plasma proteins; specifically 2-macroglubulin and C1 complement [22,23] has been shown to bind defensin. Another study [24] showed that HNP-1 bind to various plasma proteins, notably serum albumin, and it was found that serum, or serum albumin, was able to inhibit the anti-viral activity of HNP-1. This ability to bind to plasma proteins could also explain why HNP 1-3 lysis of mammalian cells is hindered in the presence of serum [25]. Together these observations add to the growing realisation that common plasma proteins may carry disease specific peptides and therefore should not be ignored in biomarker research.

Common to beta-defensin 2 (another member of the defensin family) and HNP 1-3 is an uneven distribution of surface charges. Beta-defensin 2 has been shown to bind to a chemokine receptor [26]. It has been suggested that the positively charged cluster found in defensin peptides and chemokines, may play a common role in binding to receptors, but is not important for determining receptor specificity [27]. This surface charge may also explain the binding of HNP 1-3 to plasma proteins. The

Size exclusion study of HNP 1-3 Figure 3 Size exclusion study of HNP 1-3. Protein extract from tumor tissue was separated on a peptide gelfiltration column. The elution volumes of forty (unidentified) peptides was plotted against their respective mass values and an approximate elution curve was calculated. The arrows point to HNP 1-3, which were eluted in two fractions: primarily in the void volume (8 ml) together with high mass proteins (above 20 kDa) and after 14 ml together with peptides of similar mass range (2-4 kDa). We interpret this as evidence for binding between HNP 1-3 and high mass proteins.

observation that defensins are localised to lymphocyte nuclei [28], could similarly be explained by unspecific binding to shuttle proteins.

The function of HNP 1-3

The exact concentration of HNPs in the tumour microenvironment may decide the in vivo function of HNP 1-3. One study showed that HNP 1-3 mediates lysis of tumours in a concentration dependent manner [25]. This is in agreement with another study that show that only rel-atively high concentrations of HNP-1 (10 -4 M) are cytotoxic for human monocytes, whereas lower concentration of HNP-1 (10 -8 to 10 -9 M) increases TNF-alpha production by monocytes [29]. In a study of renal cell carcinoma lines [14] it was shown that HNP 1-3 were cytotoxic to all tested cell lines when present in high concentrations (above 25 ug/ml), but at lower concentration HNP 1-3 stimulated growth of a subset of tumour cell lines. We add to these results by demonstrating that in vivo HNP 1-3 purified from colon tumours are capable of lysing MDCK cells.

Functional study of HNP 1-3 Figure 4 Functional study of HNP 1-3. Normal microscopy (A&B) and flourescence microscopy (C&D) of MDCK cells. MDCK cells were exposed to calcein with (A&C) and without HNP 1-3 (B&D). By fluorescence microscopy (C&D) the cells were observed to uptake calcein only when treated with fractions containing HNP 1-3/calcein (C). Fractions containing unidentified peptides purified from colon tumors were used as negative controls together with calcein and did not stimulate the cells to uptake calcein (D). Cell islands treated with HNP 1-3 appeared diffuse and showed enlarged nuclei, indicating apoptosis (A).

Our study was based on a 60 minutes microflow study. This screening set-up did not allow us to investigate the minimum concentration of HNP 1-3 necessary for lysis.

Conclusions

The high concentration of HNP 1-3 observed in tumours and the observation that HNP 1-3 are capable of lysing mammalian cells may lead to the conclusion that the peptides serve to the benefit of the host by killing tumour cells. However, in one study HNP 1-3 were found to bind to HLA-Class II molecules and were capable of reducing the proliferation of a HLA-DR-restricted T-cell line after stimulation [30] and could in this way help the cancer cells avoid local immune recognition. Defensins also regulate the systemic immune response. Through interaction with the chemokine receptor CCR6, beta-defensins recruits dendritic and T cells [26] (discussed in [31]) and HNP 1-3 are capable of recruiting leukocytes to sites of infection in mice [32]. Upregulated immune responses are known to stimulate tumour proliferation; immune cells are actively recruited by tumours to exploit their proangiogenic and pro-metastatic effects (reviewed in [33,34]). Whether the high concentrations of HNP 1-3 in the tumour limits the tumour growth or on the contrary stimulate tumour proliferation is not clarified; we emphasise that HNP 1-3 are expressed in inflammatory bowel disease and could be involved in the early stages of carcinogenesis. We suggest that the prominent surface charge on defensins, their unspecific binding to other proteins (such as high mass plasma proteins) and the observed excess amounts of peptides found in tumours, could provide the peptides with broad antagonising effects on numerous receptors in the tumour microenvironment. In this way HNP 1-3 peptides may serve to the benefit of the tumour.

Our results add to the evidence that HNP 1-3 bind to high mass plasma proteins. We suggest that the peptides are released in the inflammatory site and passively diffuse into the blood stream. HNP 1-3 have been observed in primary tumours of different tissues and the peptides are known to play a fundamental role in the innate immune system. We suggest that the peptides may serve as markers for colon cancer in combination with established diagnostic tools and as prognostic markers following therapy.
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Competing interestsThis study was partly financed by Colotech ltd. and partly by Glostrup Hospital. Jakob Albrethsen, Rikke Bøgebo and Hans Raskov receive salary from Colotech A/S. Steen Gammeltoft, Jesper Olsen and Benny Winther receive salary from Glostrup Hospital.Authors' contributionsJA performed the SELDI-TOF/MS screenings, the protein identification experiments and the size exclusion study. RB did the statistical analysis, BW did the microflow study, JO obtained the biological samples. HR and SG planned the project.
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A Letter Open Access Actinomycosis (Lumpy Jaw) in Capra Hircus Buck: A Case Report
2018. 2018

Mughees Aizaz Alvi
Department of Clinical Medicine and Surgery
University of Agriculture
FaisalabadPakistan

Imaad Rashid
Department of Clinical Medicine and Surgery
University of Agriculture
FaisalabadPakistan

Khurram Ashfaq
Department of Clinical Medicine and Surgery
University of Agriculture
FaisalabadPakistan

Muhammad Umar Ijaz
Department of Zoology, Fisheries and Wildlife
University of Agriculture
FaisalabadPakistan

Muhammad Haleem Tayyab
Department of Clinical Medicine and Surgery
University of Agriculture
FaisalabadPakistan

Noman Yousaf Faridi
Livestock and Dairy Development Department
Government of Punjab
Pakistan

A Letter Open Access Actinomycosis (Lumpy Jaw) in Capra Hircus Buck: A Case Report

International Journal of Molecular Veterinary Research
812018. 2018Received: 24 Nov., 2017 Accepted: 12 Jan., 20181ActinomycosisLumpy jawGoatPotassium iodied
Actinomycosis is an emerging and unanticipated infectious non-contagious disease caused by Actinomyces species affecting both humans and domesticated animals. The bacterium is opportunistic but harmless commensals of orophyranx and causes typical mandibular osteomyelitis due to which the disease is also called as 'lumpy jaw'. Different treatment protocols adopted to cure the disease have varying degree of success and time length. In this short communication, a case of actinomycosis in Capra hirus (buck) has been diagnosed and response to antibacterial therapy (potassium iodide) has been documented. It was found that potassium iodied at the rate of 70 mg/kg body weight per oral is highly efficacious for rapid recovery from the illness as compared to already evaluated antibiotics.

Background

Actinomycosis is an infectious non-contagious disease caused by Actinomyces species which are opportunistic but harmless commensals of orophyranx, lower gastrointestinal system and female reproductive tract of small and large ruminants (Moniruddin et al., 2010). The organism belongs to family Actinomycetaceae. To date a total of 42 different clinically important species of the microbe have been identified. Etiological agent is Gram-positive, non-spore forming, non-acid fast, facultative anaerobe with filamentous morphology (Reichenbach et al., 2009). The disease is likely to occur when there is breach in the continuity of mucosal layers followed by any injury, trauma or surgical course. Due to either of these predisposing factors, Actinomyces spp. penetrate into the deeper tissue and cause the clinical disease which is characterized by granulomatous abscessation which tend to be chronic in nature and lead to tissue fibrosis (Steininger and Willinger, 2016).

Pathogen can affect wide range of animals including caprines, ovines, bovines and equines. In ruminants, Actinomycesbovis causes typical mandibular osteomyelitis due to which the disease is also called as 'lumpy jaw' (Chandratre et al., 2017). The disease with abscess formation was reported for the first time in cattle in 1945 that involved jaw. The malady is an uncommon disease without any limitation of age group, sex, and breed or immunocompetency status. Actinomyces spp. has zoonotic implications as well and cause abcessation in thoracic, abdominal and cervicofacial regions of the body in human patients (Garner et al., 2007). Abscess is usually polymicrobial containing 2-4 different types of bacteria and characteristics sulphur granules. Presence of these granules depicts tissue reactions to the infection (Westhoff, 2007). Treatment regimen adopted for curing the disease generally involve long-term administration having variable cure rates and use of antibiotics for longer periods of time pave the ways for antibiotic resistance development.In this case, non-antibiotic treatment using potassium iodied was evaluated.

Materials and Methods

A 4 months old buck of was presented to the Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan on 07/07/2017 with presenting complaint of being off-fed and swelling under the jaw. Buck was examined physically. All the vitals including temperature (103.4˚F), respiration rate (18 breaths International Journal of Molecular Veterinary Research 2018, Vol.8, No.1, 1-3 http://ijmvr.biopublisher.ca 2 per minute), heart rate (75 beats per minute) and hydration status were found to be normal. There was no swelling of major lymph nodes. The only abnormality observed was presence of a mass under the mandible on left side ( Figure 1). History revealed that the mass is gradually increasing in size and animal faces difficulty while chewing the feed. Upon palpation, hard mass was felt which was immoveable. Aseptic aspiration was done and pus was oozed out of the mass. After getting complete history, observing clinical signs and conducting comprehensive physical and clinical examination, the case was diagnosed as Lumpy Jaw Disease. Animal was weighed and given potassium iodide (KI) @ dose rate of 70 mg/kg body weight orally once in a day. Total dose calculated was 1 gram per day and the treatment was continued for 25 days.

Results

Owner was also asked to pay a follow up visit so that the efficacy of treatment and its response can be evaluated. The results were found to be effective as the lesion started to regress after 10 days and completely receded after 25th day of the treatment.

Discussions

Actinomycosis is reported from all over the world but prevalence is higher in the countries with poor dental health and low socioeconomic status countries (Moniruddin et al., 2010). The disease is sporadic in its nature but causes substantial decrease in productivity of the animal aspatients' feed intake becomes decreased due to dysphagia and quidding. Facial distortion, loosening of teeth and dyspnea due to respiratory tract involvement are some of the complications of lumpy jaw. The differential diagnosis of disease includes stomatitis, rabies, fracture of mandible and tooth root abscesses (Radostits et al., 2005).

There are different treatment protocols and each of them has its own pros and cons. There has been great debate over the ideal possible therapy regimens for the cases of actinomycosis. As in lumpy jaw, abscess is mostly polymicrobial, thus no one single antibiotic can be regarded as drug of choice (Tanaka-Bandoh et al., 1997). Before the advent of penicillins and sulfa drugs, the disease led to severe disfiguring and disability in both humans and animals. With the advancements in chemotherapeutic approaches and knowledge about the pathology, both the severity and incidence of disease decreased. Almost all the antibiotics available today have variable degree of International Journal of Molecular Veterinary Research 2018, Vol.8, No.1, 1-3 http://ijmvr.biopublisher.ca 3 inhibitory effect on Actinomyces species. Antimicrobial therapy has dubious role in the cases of lumpy jaw because of questionable treatment period length and possible side effects. For example, streptomycin had been used occasionally in past but now ban has been imposed due to risk of damage to cochleovestibular system. Similarly, it has been an established fact that chloramphenicol causes bone marrow depression so its use is also limited. Actinomyces species are highly susceptible to isoniazid but the dose is 5 times more than that of which is used in tuberculosis cases. Moreover, it also poses risk of development of neurological signs. Sulfadiazine, being bacteriostatic in action, has proven efficacy but the treatment time is highly variable even leading to months or so. Moreover, it can lead to crystalluria as well in dehydrated animals (Bennhoff, 1984). Surgical debridement and surgical removal of the mass are one of the best options until and unless it is not adhere to neighboring tissue. If the lesions have involved surrounding structures and complete excision is not possible, drainage of pus followed by washing with phenol and packing with tincture iodine soaked gauze has been found useful (Sudhakar and Ross, 2004).

All of the above discussed treatment regimens are either invasive or costly enough for the small-scale farmer due to length of treatment of duration. A better and effective alternative was evaluated in this particular case. Antibacterial therapy with potassium iodide was done and treatment was continued for 25 days. Favorable prognosis was observed without any noticeable side effect as the lesion started to regress after 10 days and completely disappeared after 25 th day of the treatment.

Figure 1
1Significant swelling of mandible
Authors' contributionsMughees Aizaz Alvi, Imaad Rashid and Khurram Ashfaq were amongst the veterinary physician team at the Veterinary Clinic when the case was presented. Furthermore, Mughees Aizaz Alvi, Muhammad Umar Ijaz, Muhammad Haleem Tayyab and Noman Yousaf Faridi prepared the manuscript and final check was carried out by Mughees Aizaz Alvi.
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