Naomi Trengove

Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors

To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 μg MMP Eq/ml) compared to acute wounds (median 0.76 μg MMP Eq/ml) (p < 0.001). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase‐9 with smaller elevations of matrix metalloproteinase‐2. In addition tissue inhibitor of metalloproteinase‐1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase‐1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = – 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0.001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.

Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers

The cause of impaired healing in chronic leg ulcers is not known. However, recent attempts to modify the healing process have focused on adding growth factors to stimulate healing and have failed to produce dramatic improvements in healing. This study used a unique model of chronic wound healing in humans to obtain wound fluid samples from chronic venous leg ulcers that had changed from a nonhealing to a healing phase. These samples were used to assess cytokine and growth factor levels, and mitogenic activity in these nonhealing and healing chronic wounds. The pro‐inflammatory cytokines interleukin‐1, interleukin‐6 and tumor necrosis factor‐αwere found to be present in significantly higher concentrations in wound fluid from nonhealing compared to healing leg ulcers. There were detectable levels but, no significant change in the levels of platelet derived growth factor, epidermal growth factor, basic fibroblast growth factor or transforming growth factor‐βas ulcers healed. Wound fluid was added to fibroblasts in vitro to assess mitogenic activity. There was a significantly greater proliferative response to healing wound fluid samples compared to nonhealing samples. These results suggest that healing may be impaired by inflammatory mediators rather than inhibited by a deficiency of growth factors in these chronic wounds.

Biochemical analysis of wound fluid from nonhealing and healing chronic leg ulcers

The purpose of this study was to determine the biochemical composition of fluid taken from chronic wounds, to compare these values with that of serum, and therefore to assess whether the wound fluid is representative of the extracellular environment of the wound. Paired wound fluid and blood samples were collected from eight patients with chronic leg ulcers in a nonhealing and healing phase. Wound fluid and serum samples were screened for a profile of general biochemical analyses, including electrolytes, lactate, glucose, and protein analyses. Electrolyte levels were essentially identical in wound fluid and serum samples. Lactate and lactate dehydrogenase levels were significantly greater and glucose and bicarbonate levels were significantly lower in wound fluid when compared with the paired serum samples. Albumin and total protein levels in wound fluid were on average half those of serum levels. In this small sample of eight patients, wound fluid collected from chronic leg ulcers is an exudate with the biochemical composition expected in extracellular fluid. In addition, bicarbonate and glucose levels increase and C‐reactive protein levels decrease in wound fluid, but remain unchanged in serum, during healing. These results suggest changes in the state of hypoxia and the inflammatory process in the healing wound.

Breastmilk is a novel source of stem cells with multilineage differentiation potential

The mammary gland undergoes significant remodeling during pregnancy and lactation, which is fuelled by controlled mammary stem cell (MaSC) proliferation. The scarcity of human lactating breast tissue specimens and the low numbers and quiescent state of MaSCs in the resting breast have hindered understanding of both normal MaSC dynamics and the molecular determinants that drive their aberrant self‐renewal in breast cancer. Here, we demonstrate that human breastmilk contains stem cells (hBSCs) with multilineage properties. Breastmilk cells from different donors displayed variable expression of pluripotency genes normally found in human embryonic stem cells (hESCs). These genes included the transcription factors (TFs) OCT4, SOX2, NANOG, known to constitute the core self‐renewal circuitry of hESCs. When cultured in the presence of mouse embryonic feeder fibroblasts, a population of hBSCs exhibited an encapsulated ESC‐like colony morphology and phenotype and could be passaged in secondary and tertiary clonogenic cultures. While self‐renewal TFs were found silenced in the normal resting epithelium, they were dramatically upregulated in breastmilk cells cultured in 3D spheroid conditions. Furthermore, hBSCs differentiated in vitro into cell lineages from all three germ layers. These findings provide evidence that breastmilk represents a novel and noninvasive source of patient‐specific stem cells with multilineage potential and establish a method for expansion of these cells in culture. They also highlight the potential of these cells to be used as novel models to understand adult stem cell plasticity and breast cancer, with potential use in bioengineering and tissue regeneration. STEM Cells2012;30:2164–2174

Maternal and infant infections stimulate a rapid leukocyte response in breastmilk

Breastmilk protects infants against infections; however, specific responses of breastmilk immune factors to different infections of either the mother or the infant are not well understood. Here, we examined the baseline range of breastmilk leukocytes and immunomodulatory biomolecules in healthy mother/infant dyads and how they are influenced by infections of the dyad. Consistent with a greater immunological need in the early postpartum period, colostrum contained considerable numbers of leukocytes (13–70% out of total cells) and high levels of immunoglobulins and lactoferrin. Within the first 1–2 weeks postpartum, leukocyte numbers decreased significantly to a low baseline level in mature breastmilk (0–2%) (P<0.001). This baseline level was maintained throughout lactation unless the mother and/or her infant became infected, when leukocyte numbers significantly increased up to 94% leukocytes out of total cells (P<0.001). Upon recovery from the infection, baseline values were restored. The strong leukocyte response to infection was accompanied by a more variable humoral immune response. Exclusive breastfeeding was associated with a greater baseline level of leukocytes in mature breastmilk. Collectively, our results suggest a strong association between the health status of the mother/infant dyad and breastmilk leukocyte levels. This could be used as a diagnostic tool for assessment of the health status of the lactating breast as well as the breastfeeding mother and infant.

Variation in fat, lactose, and protein composition in breast milk over 24 hours associations with infant feeding patterns

Background: Data regarding the association between breast milk composition and infant feeding patterns (frequency and amount of breast milk taken) would help in understanding the regulation of food intake in breastfed infants. Objective: This study examined the relationship between breast milk macronutrient concentration and patterns of milk intake in breastfeeding infants over a 24-hour breastfeeding period. Methods: Mothers of healthy term infants (n = 15) collected pre- and postfeed breast milk samples from each feed at each breast over a 24-hour period. Breast milk samples were analyzed for fat, lactose, total protein, casein, and whey protein content. The energy content for each feed was calculated. Results: Breastfeeding patterns and milk composition varied greatly between individuals. The fat content of milk significantly differed over 24 hours (P = .01), whereas the concentration of lactose and protein content remained the same. The mean 24-hour total protein, whey, and casein intake was inversely (P < .01), whereas lactose concentration was positively (P = .03) related to the number of breast feeds per day. No relationship was seen either between fat or energy content and feeding patterns. The mean (SD) concentration of fat, lactose, and total protein over the 24-hour period was 43 (12) g/L, 68 (7) g/L, and 13 (2) g/L, respectively. Conclusion: The association between milk protein intake and the breastfeeding frequency suggests that the protein intake may play a role in infant appetite control.

Analysis of insulin in human breast milk in mothers with type 1 and type 2 diabetes mellitus.

Despite the important role that insulin plays in the human body, very little is known about its presence in human milk. Levels rapidly decrease during the first few days of lactation and then, unlike other serum proteins of similar size, achieve comparable levels to those in serum. Despite this, current guides for medical treatment suggest that insulin does not pass into milk, raising the question of where the insulin in milk originates. Five mothers without diabetes, 4 mothers with type 1, and 5 mothers with type 2 diabetes collected milk samples over a 24-hour period. Samples were analysed for total and endogenous insulin content and for c-peptide content. All of the insulin present in the milk of type 1 mothers was artificial, and c-peptide levels were 100x lower than in serum. This demonstrates that insulin is transported into human milk at comparable concentration to serum, suggesting an active transport mechanism. The role of insulin in milk is yet to be determined; however, there are a number of potential implications for the infant of the presence of artificial insulins in milk.

Using milk flow rate to investigate milk ejection in the left and right breasts during simultaneous breast expression in women.

BackgroundMilk ejection is essential for a successful lactation, however techniques to measure milk ejection in women are often complex and invasive. Recent research has demonstrated that at milk ejection, milk duct diameter increased in the breast (measured by ultrasound) at the same time as milk flow rate increased (measured using a weigh balance). This study aimed to evaluate a purpose-built continuous weigh balance (Showmilk, Medela AG) to measure changes in milk flow rate from the breast to identify milk ejections during milk expression. In addition, the Showmilk was used to determine if milk ejection occurred simultaneously in both breasts during double pumping.MethodsIncreased milk flow rates during single pumping were compared to simultaneous ultrasound measurements of increased milk duct diameters in 14 mothers. In addition, increases in milk flow rate were compared between the left and right breasts of 28 mothers during double pumping for 15 minutes with two separate electric breast pumps attached to two Showmilks to record milk flow rate.ResultsIncreased milk flow rates were associated with increased milk duct diameters during single pumping. The mean number of milk ejections was not different between the Showmilk (4.2 ± 2.0) and ultrasound (4.5 ± 1.5) techniques. Overall, 67 milk ejections were measured and of these, 48 (72%) were identified by both techniques. The left and right breasts responded synchronously with 95.5% of the flow rate increases corresponding between the breasts. The mean number of milk ejections identified by an increase in milk flow rate during double pumping was 5.1 ± 1.7 and 5.0 ± 1.7 for the left and right breasts, respectively. In addition, mothers chose the same expression vacuum for the left (-198 ± 31 mmHg) and right (193 ± 33 mmHg) breasts.ConclusionThe Showmilk can simply and non-invasively record milk ejections by measuring increases in milk flow rate that correspond with increases in milk duct diameter. For the first time measurement of milk flow rate has been used to confirm that milk ejections occur simultaneously in the left and right breasts during double pumping. The use of the Showmilk will facilitate further research into the relationship of milk ejection and milk removal.

Investigation of Short-term Variations in Term Breast Milk Composition during Repeated Breast Expression Sessions

Background: Breast milk composition can be affected by several factors, and it can exhibit short-term (weekly) variations. Investigating variations in breast milk composition is important to accurately estimate nutrient requirements of the infant. Objective: To investigate short-term changes in breast milk composition between left and right breasts, over a 3-week period within the first 6 months of lactation. Methods: The left and right breasts of the mothers of healthy, term infants (n = 23) were simultaneously expressed with an electric breast pump for 15 minutes, on 3 occasions within 3 weeks. Milk samples (5 mL) were collected from the total expression volume of each breast at each session. The macronutrient contents, total solids, and energy content were determined using a mid-infrared human milk analyzer. Mothers (n = 17) measured their 24-hour milk production, and the average 24-hour fat contents were also determined. Results: Over the 3 weekly sessions, no significant changes were found in macronutrient contents. On average, total solids (P = .04) and energy (P = .04) decreased by week 3 of follow-up sessions from 14 to 13 g/100 mL and from 82 to 76 Kcal/100 mL, respectively; however, these changes became insignificant when expression volume was taken into account. The macronutrient concentration was similar for the left and right breasts; however, milk composition varied markedly between mothers. Furthermore, average 24-hour fat content was significantly lower than the mean fat content from a single expression session (P < .01). Conclusion: Our findings highlight that when determining the nutritional adequacy of a mother’s milk, assuming an average concentration requires caution. The study findings illustrate the importance of using average 24-hour fat content of milk to obtain representative measures of infant energy intake.

Comparison of the patterns of milk ejection during repeated breast expression sessions in women.

BACKGROUND This study aimed to investigate the consistency of milk ejections and milk expression characteristics within mothers at repeated expression sessions. METHODS Twenty-five breastfeeding mothers expressed their breasts simultaneously on three occasions within 3 weeks, and follow-up visits were performed at 6, 9, and 12 months of lactation. During the 15-minute expression, milk was collected onto a continuous weigh balance to measure milk flow rate. RESULTS The number of milk ejections was similar at the three sessions (5.1±2.0), decreasing at the 12-month follow-up (3.3±1.2). Mothers had a similar pattern of milk ejection at each session. The time that each milk ejection occurred was consistent for the first 9 months of lactation. Of the four milk ejection patterns identified, each removed a similar percentage of available milk but varied in the time to reach 80% of the total expression volume. The first two milk ejections produced the greatest percentage (62%) of total milk volume during breast expression. CONCLUSIONS For each individual mother, the timing, pattern, and number of milk ejections were consistent, suggesting a predetermined release of oxytocin. In light of the innate oxytocin release and milk removal characteristics in women, there is potential for individual tailoring of the duration of expression.