CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/675,143, filed July 24, 2012 and U.S. Provisional Application No. 61/730,555, filed November 28, 2012, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with Government support under grant numbers R0 HD059142 and 2U10HD034216-16 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Although its etiology is unclear, NEC is associated with various pre- and postnatal factors, including placental insufficiency, prolonged premature rupture of membranes, chorioamnionitis, gut ischemia, altered bacterial colonization, viral infections of the gastrointestinal tract, and blood transfusions. These conditions presumably alter and/or disrupt the gut epithelial barrier and promote the translocation of luminal bacteria, triggering a damaging inflammatory reaction.

Regarding NEC, several cross-sectional studies have shown increased expression of inflammatory cytokines such as tumor necrosis factor (TNF), interleukins (IL), including IL-Ι β, IL-6, IL-8/CXC-motif ligand 8 (CXCL8) in both affected tissues and plasma [1-4]. Cytokines are potentially-attractive therapeutic targets in NEC because many of them, including TNF, IL-Ιβ, and IL-8/CXCL8, can disrupt the epithelial barrier and augment tissue damage in diverse forms of neonatal intestinal injury, and because of the availability of monoclonal antibodies and/or small molecule inhibitors against these inflammatory mediators. At the same time, concerns remain about possible harm from anti-cytokine therapy in preterm infants because many of the so-called 'inflammatory' cytokines, including IL-8 and TNF, play a key role in the development of the crypt-villus architecture and mucosa-associated immune system. Other cytokines, such as transforming growth factor-beta (TGF-β), play a role in maintaining the normal absence of inflammation in the intestinal mucosa despite close physical proximity to the gut luminal flora [5]. However, understanding the role of cytokines in NEC from cross-sectional studies has important limitations because cytokine expression changes significantly during normal gestational maturation and also with co-morbidities associated with prematurity [6-8]. Furthermore, such studies are also likely to miss pre-existing conditions such as a fetal inflammatory response.

There is a need in the art for improved methods of predicting risk of an infant for developing NEC. The present invention addresses that need.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention includes methods of predicting the risk of an infant for developing NEC by measuring the concentration of circulating TGF- β in a blood sample from the infant. In certain embodiments, the concentration of TGF-β in a blood sample from the infant is compared to a reference, for example, the concentrations of TGF-β in the blood of populations of infants with and without NEC. A low blood concentration of TGF-β relative to a population of infants that do not have NEC indicates that the infant has an increased risk or a relatively high risk of developing NEC compared to an infant whose blood concentration of TGF-β is not low relative to a population of infants that do not have NEC. In certain embodiments, a TGF-β concentration of less than 1380 pg/mL is predictive of increased risk of developing NEC.

In certain embodiments, the infant is premature, i.e., born prior to 40 weeks gestation. In certain embodiments, the infant has a low birth weight. In certain embodiments, the infant has a birth weight of 1000 grams or less. In certain embodiments, the infant is asymptomatic, i.e., the method of the invention is performed on an infant prior to development of symptoms of NEC.

In certain embodiments, the blood sample is a blood spot collected on filter paper. In certain embodiments, the blood sample is an eluate of a blood spot. In other embodiments, the blood sample may be collected from a heel stick using a capillary tube with or without an anticoagulant. In other embodiments, the blood sample may be collected by venipuncture in the presence or absence of an anticoagulant. In certain embodiments, the assay may be performed on whole blood, plasma, or serum.

In certain embodiments, TGF-β measurements may be used in conjunction with other observations associated with NEC, including, but not limited to, the absence or reversal of umbilical blood flow on prenatal Doppler examination and/or the detection of villitis or vasculitis on placental histopathology. Advantageously, the use of various markers in combination may improve the ability to predict NEC in premature infants.

It is an advantage that certain embodiments of the method of the present invention may improve the identification of infants at increased risk for developing NEC. In certain embodiments, identification of an infant at increased risk for developing NEC further involves selective application of technology-based interventions such as intensive monitoring of splanchnic perfusion and oxygenation, devices that are potentially-useful but are expensive or cumbersome for universal use in the neonatal intensive care unit. Such an approach could also help direct interventions such as the use of banked human milk and/or probiotics, which are promising but of unproven safety in low birth weight infants. The availability of a test to predict NEC has the potential to change clinical practice and reduce morbidity and mortality associated with this condition.

In a further embodiment, this invention is a method of diagnosing necrotizing enterocolitis in an infant comprising performing an assay on a sample of blood from the infant to determine the level of TGF-β concentration in the blood wherein a measured concentration of TGF-β lower than the TGF-β concentrations of a population of infants that do not have NEC is diagnostic of NEC. In certain embodiments, a TGF-β concentration that is lower than about 1380 pg/mL is diagnostic of necrotizing enterocolitis.

The present invention and its attributes and advantages will be further understood and appreciated with reference to the detailed description below of presently contemplated embodiments, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

The preferred embodiments of the invention will be described in conjunction with the appended drawings provided to illustrate and not to the limit the invention, where like designations denote like elements, and in which:

FIG. 1 is a receiver-operating characteristic (ROC) plot of true positives vs. false positives at a TGF-β threshold of 1380 pg/mL; and

FIG. 2 illustrates a method embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Longitudinal changes in cytokine expression in premature infants starting at birth and prior to onset of NEC were investigated by performing a secondary analysis of data obtained as part of the National Institute of Child Health and Human Development (NICHD) Cytokine Study, a prospective multi-center study in which ELBW infants, i.e., infants having a birth weight of 1000 grams or less, were enrolled and clinical information and serial cytokine measurements were collected from birth through postnatal day 21 [6-8].

Preterm neonates with birth weight 401-1000 g were enrolled after obtaining written informed consent from the parent(s). Whole blood spots were collected on standardized filter paper and frozen on postnatal days 0-1 (D1 ), 3±1 (D3), 7±1 (D7), 14±3 (D14), and 21 ±3 (D21 ) using an established protocol that has been shown to maintain sample quality and consistency for cytokine measurements over extended periods of time (>20 years) [9]. Clinical data were collected by trained research coordinators and analyzed at a central data coordinating center. Concentrations of 25 cytokines/inflammatory mediators were measured in blood spot eluates using a multiplex flow cytometric immunoassay based on laser detection of color-encoded antibody-tagged microspheres (xMAP assay, Luminex Corp., Austin, TX) [9, 10]. The following analytes were included in the immunoassay panel: IL-Ι β, IL-2, IL-4, IL-5, IL-6, IL-12, IL-17, IL-18, TNF-a, lymphotoxin-alpha (LT-a)/TNF-beta, interferon (IFN)- Y, granulocyte macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein (MCP)-1/CC-motif chemokine ligand (CCL)-2, macrophage inflammatory protein (MIP)-1a/CCL3, MIP-i /CCL4, regulated on activation, normal T-cell expressed and secreted (RANTES)/CCL5, IL-8/CXCL8, IL-10, TGF-β-ι, matrix metalloproteinase (MMP)-9, soluble IL-6 receptor (slL6R), triggering receptor expressed on myeloid cells (TREM)-1 , brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and C-reactive protein (CRP). This assay has low intra- (<10%) and inter-assay (7-23%) variation and has lower limits of detection lower than reported median plasma concentrations of these cytokines/inflammatory factors in normal neonates.

In the present analysis, all preterm neonates enrolled in the NICHD cytokine study with a history of NEC (Bell's stages II and III) [11 , 12] were included. All other infants were included in the control group. Demographic and clinical data, including gestational age, birth weight, gender, ethnicity, history of sepsis, and postnatal age at onset of NEC were obtained from the NRN generic database. Frequencies (percentage), means, standard deviations, median values, and ranges were computed for demographic data and cytokine concentrations. Frequencies were compared by the Fisher's exact test [13]. All cytokine concentrations were rounded to the nearest picogram, the least significant figure for the immunoassay.

First, peak cytokine concentrations in infants in the NEC and control groups were compared. Then, to determine whether changes in cytokine expression preceded the clinical diagnosis of NEC, samples from infants in the NEC group drawn prior to onset of NEC were compared to samples from the control infants. Finally, to determine the change in cytokine levels due to the onset of NEC, each sample from the NEC group were classified by the day when it was drawn and into a 'before NEC or an 'after NEC group. All group-wise comparisons of cytokine concentrations were performed using the Student's t test (for groups with equal variance [14] or the Welch-Satterthwaite f-test (when variance was unequal) [15].

To identify a 'cut-off TGF-β level prior to onset of NEC that could discriminate between infants who developed NEC vs. controls, a receiver operating characteristic (ROC) curve was constructed by plotting sensitivity vs. 1 - specificity for various TGF- β levels [16]. The diagnostic accuracy of the cut-off value was computed using a 2x2 confusion matrix with the predicted vs. observed number of cases and the number of true and false positives were calculated [17]. To identify other clinical and cytokine predictors of NEC, a stepwise logistic regression analysis was performed. Univariate logistic regression was first used to evaluate categorical predictors such as gender (1 = male, 2 = female), ethnicity (1 = African-American, 2 = Caucasian, 3 = other), and sepsis (1 = yes, 0 = no), and then continuous predictors such as gestational age, birth weight, and mean cytokine values from D1 , D3, D7, D14, and D21. Entry and stay criteria for each variable were set to p = 0.20. Variables identified in univariate analyses at p<0.20 level (and other variables not selected at this cutoff but considered clinically important) were used to develop a multivariate survival model [18, 19]. Cytokines were measured at serial time-points, and were therefore, considered time-dependent, right-censored (because many infants developed NEC beyond D21) covariates [20]. For categorical predictors, Kaplan- Meier curves were examined and then the log-rank test for equality across strata [21] was used to identify predictors (p<0.25) to be included in the final model. For continuous variables, a univariate Cox proportional hazard regression model was used [22], and a significant Chi-squared statistic (p<0.25) was accepted as an indicator for an association with NEC. Variables identified in logistic regression were tested sequentially to determine whether one/more of these variables in combination with TGF-β could predict NEC better than TGF- β alone.

Patient characteristics: A total of 1067 infants with a birth weight between 410-1000 g were admitted to the participating centers during the study period. Infants were enrolled within 72 hours after admission, although all infants were not able to complete the full duration of the study. Of the 574 infants enrolled on D1 , 358 completed the study. The others were not able to complete the full duration of the study due to transfer to other hospitals, withdrawal from the study or death. Similarly, 434 neonates were enrolled on D3, 344 of which completed the study. In the final analysis, a total of 997 infants were included. Seventy neonates were excluded from the study due to death in the first 7 days (n = 43), birth defects (n = 13), or early- onset sepsis (n = 16).

NEC was recorded in 104/997 (10.4%) infants with a median age of onset of 23.5 days (range 1-114 days). Demographic characteristics of the NEC and control groups are summarized in Table 1. The two groups had a similar distribution of gestational age, birth weight, and gender (p-values > 0.05). Consistent with existing information [23, 24], the NEC group had a higher percentage of African-American infants (61/104, 58.6% in the NEC group vs. 423/997, 47.3% controls, p = 0.02) and also had a higher incidence of sepsis (52.9 vs. 41.6%, p = 0.02). Table 1. Demographic and Clinical Information

^Total number of controls with complete information varies by variable.

'Statistical significant differences at 5% level.

Cytokine concentrations (unadjusted): As noted previously, time trends (p<0.05) were identified during the first 3 weeks for all cytokines, which remained significant even after controlling for center, gestational age, gender, ethnicity, late-onset sepsis, antenatal steroids, postnatal steroids, days in oxygen, severe intra-ventricular hemorrhage, and persistent patency of the ductus arteriosus treated with surgery. IL- 1 p, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, LT-a, NT4, and GM-CSF decreased with time, whereas IL-18, BDNF, MMP-9, RANTES/CCL5, TGF-β, slL-6R, and CRP increased with postnatal age.

Infants who developed NEC were discovered to have lower TGF-β levels than controls (median 1871 , range 467-11431 pg/mL vs. 2501 , range 202-12750 pg/mL, p<0.05). There was also a trend towards lower IL-2 concentrations in the NEC group. In contrast, infants in the NEC group had higher IL-8/CXCL8 levels than controls. These data are presented in Table 2. Table 2. Summary statistics for peak cytokine concentrations in cases and

Peak calculated as the Maximum Cytokine for each infant.

For Cases: Peak calculated using all sample days

For Controls: Peak calculated using all sample

days

Testing the average values, test is a two sample t- test

Cytokine concentrations in cases prior to onset of NEC: To determine whether the differential expression of cytokines detected in NEC vs. control groups antedated or followed the onset of NEC, samples from infants in the NEC group drawn prior to onset of NEC were compared to samples from control infants. Infants in the NEC group showed lower median TGF-β levels than controls on all sample days (D1 : median 1090, range 50-7305 pg/mL in cases vs. 1301 , 56-7380 pg/mL; D3: 988, 50-6869 vs. 1286, 0- 7095 pg/mL; D7: 1073, 50-5364 vs. 1487, 9-6978 pg/mL; D14: 1573, 50-5677 vs. 1952, 50-10022 pg/mL; D21 : 1701 , 98-1 1431 vs. 2129, 50-12750 pg/mL; p<0.05 for all subgroup comparisons). The NEC group also had lower BDNF on D1 , lower IL-18, MIP- 1a/CCL3, RANTES/CCL5, and MMP-9 on D3, lower IP-1a/CCL3, and RANTES/CCL5 on D7, lower IL-2 and IL-4 on D14, and lower IL-2, IL-17, BDNF, and NT-4 levels on D21. The longitudinal change in median cytokine concentrations in these two groups is shown in Table 3.

Table 3. Summary statistics for in cytokine concentrations in cases prior to onset of NEC vs. controls summarized by day of measurement

TGF-β as a predictor of NEC: In logistic regression analysis, TGF-β alone was a better predictor of NEC (p<0.001) than in combination with clinical characteristics or other cytokines. To develop a test to discriminate between infants who went on to develop NEC vs. others who did not, an ROC curve was produced using TGF-β concentrations from all cases before onset of NEC and controls (FIG 1; Table 4). The area under the curve (AUC) was 0.67, indicating "fair" diagnostic accuracy. The use of cumulative TGF-β increased the AUC to 0.71. Because a single, random measurement is the most feasible approach in the clinical setting, a cut-off TGF-β value that would diagnose at-risk infants was sought. A cut-off value of TGF-β <1380 pg/mL on any sample day classified infants into the NEC or control group with an accuracy of 64.0% (95% confidence interval 60.9% - 66.9%). The proportion of true positives (sensitivity) and true negatives (specificity) was 61.2% and 64.3%, respectively.

Table 4

Low circulating TGF-β has been identified as a novel biomarker for NEC in low birth weight infants. In blood spot eluates, TGF-β concentrations of less than 1380 pg/mL predicted NEC with about 64% accuracy. This is believed to be the first plasma biomarker that can help estimate the risk of NEC in an asymptomatic premature infant. Infants who develop NEC have been discovered to have lower TGF-β levels from D1 , indicating that the factors that increase the risk of NEC appear early, most likely in utero.

While the present invention has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the present invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from its scope. Therefore, it is intended that the present invention not be limited to the particular embodiments or examples disclosed, but that the present invention will include all embodiments falling within the scope of the appended claims.

List of publications cited

Each of the following publications is incorporated by reference in its entirety:

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