Bovine Colostrum In The Treatment Of Severe Alcohol-Associated Hepatitis

Introduction and review of literature Severe alcoholic hepatitis is associated with significant morbidity and mortality.

4.1) Diagnosis: The diagnosis of Alcoholic hepatitis is made by the following criteria 12

1. *Inclusion criteria:
2. Onset of jaundice within the prior 8 weeks
3. Ongoing consumption of > 40 (female)g or > 60 (males) g alcohol/day for 6 months or more, with less than 60 days of abstinence before the onset of jaundice
4. Aspartate aminotransferase (AST) > 50, AST/alanine aminotransferase (ALT) >1.5, and both values < 400 IU/L
5. Serum bilirubin (total) > 3.0 mg/dL
6. Maddrey's discriminant function (DF) > 32
7. Model for End-stage Liver Disease (MELD) score > 20 *Exclusion Criteria:

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1. Uncontrolled infections
2. Multiorgan failure
3. Uncontrolled upper gastrointestinal bleeding#
4. Preexisting kidney injury with serum creatinine > 2.5 mg/dL
5. Other chronic liver diseases
6. Hepatocellular carcinoma, other active malignancies, except skin cancer
7. Pregnancy
8. Uncontrolled substance use disorder
9. Known cow milk allergy or severe lactose intolerance**
10. Treatment with Corticosteroids

• Crabb DW, Bataller R, Chalasani NP, et al. Standard Definitions and Common Data Elements for Clinical Trials in Patients With Alcoholic Hepatitis: Recommendation From the NIAAA Alcoholic Hepatitis Consortia. Gastroenterology 2016;150(4):785-790.
• Patients with upper Gastrointestinal bleeding controlled with vasopressors and endoscopic haemostasis, were included in the study. ** The diagnosis of Lactose Intolerance was made clinically and confirmed by the Hydrogen Breath Test.

IX. 4.2) Assessment of Severity 4.2.1)Maddrey's Discriminant Function15 Discriminant Function Index (DFI) was originally described by Maddrey and colleagues in a placebo controlled study to assess the benefit of corticosteroids in patients with AH. The original formula was 4.6 x prothrombin time (seconds) + serum Bilirubin (mg/dl). It was observed that patients with a DFI >93 and treated with placebo had a 28 day survival of 25%, whereas those with a DFI < 93 had a 100% survival. Subsequently this score was modified in 1986 and called Modified Discriminant Function (MDF). MDF = 4.6 x (Patient's PT - Control PT) + serum bilirubin (mg/dl). Untreated patients with MDF >32 had a survival of 68%. The American College of Gastroenterology 9 recommends that patients with MDF > 32 and / or Encephalopathy should be treated. The prothrombin time varies greatly depending on the sensitivity of the thromboplastin reagent used for the test. This is the drawback of the MDF.

4.2.2) MELD score16 The model for end stage liver disease (MELD) score predicts survival in patients with cirrhosis and is used to prioritize patients for liver transplantation.

MELD score = (0.957 x log serum creatinine + 0.378 x log serum bilirubin + 1.120 x log INR + 0.643) x 10. The advantage of MELD score is the use of INR in place of PT. The INR is comparable and uniform across laboratories worldwide. The American Association for Study of Liver Diseases recommends that a MELD score >20predicts SAH and should be the criterion for initiating treatment. Serial monitoring of MELD score with a change of 2 or more points over the first week on treatment has independently predicted mortality.

4.2.3) GAHS score17 Glasgow Alcoholic Hepatitis Score (GAHS) includes age, peripheral Total Leucocyte Count, serum Bilirubin (at days 1 and 6-9), blood urea nitrogen and PT. It is a better predictor of mortality at 28 days than MDF. GAHS score > 9 has an extremely poor prognosis unless treated with Corticosteroids. The drawback is lack of international validation.

4.2.4) LILLE'S model18 It is not traditionally used for predicting mortality of SAH. However, it helps to predict mortality at 7 days of SAH patients on Corticosteroids .If the score is more then 0.45 then Corticosteroids should be stopped.

The diagnosis ofSevere Alcoholic Hepatitis is made by the following criteria

1. MDF>32, or
2. MELD>20

4.3) Treatment 4.3.1)Abstinence from alcohol This is the most important factor in predicting the outcome after surviving the acute alcoholic hepatitis (AH) episode. The incidence of recidivism after recovery from the first episode of AH varies from 10 - 70 %. Abstinence will be reinforced by giving patient oral/enteral Baclofen (GABA B receptor agonist)

4.3.2)Nutrition Enteral nutrition is the preferred mode for supplemental nutrition. It is cheap, maintains gut mucosal integrity. This in turn decreases the risk of bacterial and endotoxin translocation resulting in less infections and endotoxemia. Pooled data from 5 RCT's of AH have demonstrated improved nutritional status compared to standard dietary intake, but without improving survival rates.19 The patient will be given 30- 40kcal/kg diet along with 1.5gm/kg protiens, emphasizing breakfast and a night time snack with regular oral diet. Frequent interval feeding will be given to improved nitrogen balance. Bcomplex multivitamins supplementation will be provided to all patients (EASL guidelines14) 4.3.3) Pharmacotherapy of Severe Alcoholic Hepatitis 4.3.3.1) Corticosteroids Corticosteroids are the most extensively used treatment for Severe Alcoholic Hepatitis. 6/13 Randomized Controlled Trials and 4/6 meta-analysis have supported the use of Corticosteroids in Severe Alcoholic Hepatitis. The last meta-analysis of individual patient data from 5 high quality Randomized Controlled Trials using Corticosteroids forSevere Alcoholic Hepatitis concluded that Corticosteroids confers a 50% relative survival benefit at 1 month (85% versus 65% survival amongst those treated with Corticosteroids versus untreated patients). The number of patients needed to treatedis 5 to reduce one death. Oral Prednisolone 40mg daily or parenteral methylprednisolone (for patients unable to take orally) 32 mg intravenously daily for 4 weeks is the standard therapy. A Lille score of >0.45 after 7 days of treatment with Corticosteroids is an indication of non-response, increased susceptibility to infections and diminished survival20. The Corticosteroids should be stopped if the Lilli score is >0.45. The most recent STOPAH trial was unable to show a significant improvement in mortality of SAH patients treated with Corticosteroids. There was a trend in favour of Corticosteroids.

4.3.3.2)Pentoxifylline Pentoxifylline is a phosphodiesterase inhibitor. It is given orally in the dose of 400mg thrice a day for 28 days. In a pivotal study of 101 patients of SAH21, it was associated with survival benefit of 50%. In another study, it was found to be superior to Corticosteroids22.

In a recent study involving 50 patients at our center23, we found that 20% vs 40% mortality in patients treated with Pentoxifylline versus controls, after 4 weeks of treatment. Acute kidney injury was the cause of death in 20% compared to 70% patients in Pentoxifylline group versus controls. Pentoxifylline induces recovery of renal \& hepatic functions with a trend towards increased survival. However, a metanalysis of 5 Randomized Controlled Trials failed to show any benefit with Pentoxifylline24.In a large recent trial Corticosteriods plus Pentoxifyllinefor 4 weeks treatment with compared with prednisolone alone, did not result in improved 6-month survival.25 The major adverse effects include vomiting, diarrhea, abdominal pain, headache and skin rash.

4.3.3.3) N-Acetylcyteine N-acetylcysteine could have value as an antioxidant in the treatment of acute alcoholic hepatitis,because the thiol group in N-acetylcysteine is able to reduce levels of free radicals. Administration of N-acetylcysteine might reconstitute the glutathione stocks of the hepatocytes.

At present, N-acetylcysteine is used in the treatment of acetaminophen induced hepatitis. Intravenous acetylcysteine is most often used as an antidote for acetaminophen overdose due to its ability to increase levels of glutathione; however, it is also used to treat non-acetaminophen-induced acute liver failure (NAI-ALF) and severe alcoholic hepatitis and to prevent contrast-induced nephropathy (CIN). Although the i.v.and oral formulations of acetylcysteine have been evaluated for these indications, most studies have examined the i.v.form. I.V. acetylcysteine is used in the treatment of NAI-ALF to improve oxygenation to the liver. A trial to evaluate the efficacy of glucocorticoids plus N-acetylcysteine, as compared with glucocorticoids alone, in patients with severe acute alcoholic hepatitis.26 reported improved one month survival in the combined treatment group. However the 6 month survival was not different in the two groups.

4.3.3.4) Bovine colostrum Human colostrumis the first milk produced after birth and is particularly rich in immunoglobulin, antimicrobial peptides (lactoferrin and lacto peroxidase) and other bioactive molecule, including growth factors which are important for nutrition, growth and development of newborn infants and also for passive immunity. Bovine colostrum is produced by the cows during the first one day post parturition. This "early" milk has nutrient profile and immunological composition substantially different from 'mature' milk. It contains macronutrient like proteins, carbohydrate, oligosaccharides, fats and micronutrients like vitamins and minerals, also growth factors, anti-microbial compounds and immune regulating constituents either not present in mature milk or present substantially in lower concentration.27,28 Bovine colostrum used in the treatment of HIV-positive patients with chronic diarrhea,29androtavirus diarrhea in children.30 Available evidence suggests a beneficial effect of supplementation of bovine colostrums in improving body composition, aspects of athletic performance, diarrhea in persons with immune-deficiency syndromes, NSAID-induced gastrointestinal ulcers, and aspects of the acute phase response that occurs secondary to surgery. Specific hyperimmune bovine colostrums, produced to have high neutralizing titer activity against Cryptosporidia, H. pylori, measles, rotavirus, and Shigella sp., appear to have clinical utility in conditions associated with these infectious organisms31 Historically colostrum has been used for various illnesses in India as well as abroad for thousands of years. Ayurvedic physicians of India have used bovine colostrum for both medicinal and spiritual purposes since cows were first domesticated. By the late 18th century, Western medicine started to take an interest in colostrum and study it for its potential health benefits. As a consequence, it was prescribed for many conditions, including immune system enhancement. Interestingly, until the development of penicillin and other artificial antibiotics in the 20th century, colostrum was commonly used for fighting bacterial infections. In the early 20th century it was noted that antibody levels in the first milk produced after birth were much higher than levels found in the milk that was produced 72 hours later.There are now over 2000 published scientific articles that document the safety and efficacy of using colostrum. This seemingly perfect food has been shown to be non-species specific32. In 2005, it was reported that is safe and effective in repair of tissues as well as for enhancing immunity.33 The use of bovine colostrum as dietary supplement has increased substantially over the past decades. Bovine colostrum is harvested within first few hours of calving from dairy animals. The herds of cows are kept under close supervision in good state of hygiene without exposure to antibodies, pesticides and anthelmintics. The colostrum collected within 24 hours contains maximum substances but less in amounts, colostrum collected later will be more in quantity but contain less immunoglobins.33 Colostrum contains the growth factors that help build lean muscle, including insulin-like growth factors (IGF-I \& IGF-II) and growth hormone (GH). IGF-I, which is found naturally in colostrum, is the only natural hormone capable of promoting muscle growth by itself. The IGFs in humans and cows are identical, but bovine colostrum actually contains a greater concentration of IGF-I than human colostrum. This fact makes bovine colostrum attractive to bodybuilders, athletes and others seeking to gain muscle mass. According to a study conducted in Finland, IGF-1 induces protein synthesis, which leads to an increase in lean muscle mass without a corresponding rise in adipose (fat) tissue. The growth factors in colostrum "shift fuel utilization from carbohydrate to fat. It mean that body will burn more fat, including fat made from the carbohydrate and protein that are consumed, producing fuel more efficiently.34 Bovine has hormone factor like gastrin which plays a role as a trophic factor for mucosal growth with in stomach. GLP-2 has shown a general trophic response with in the gut.

Peptide growth factors in colostrum can influence the maintenance of mucosal mass and integrity. EGF stimulates cell proliferation and also influences crypt fission.25 Human colostrum has higher concentration of growth factors as compared to bovine colostrum. The tables and figure below indicate how HC and BC differ in their nutrient content (9,35,36)

While BC are considered a rich source of IgG, growth factors, and lactoferrin, currently no specific standards exist that define BC dietary supplements with respect to actual constituents or amounts of constituents. The following constituents are generally present in bovine colostrums: macronutrients; vitamins; minerals; Ig (IgG, IgM, and IgA - including the secretory form); cytokines including interleukin-1beta (IL-1β), interleukin- 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (INF-γ); growth factors including insulin-like growth factor (IGF) I and II, transforming growth factor-beta (TGF-β), and epidermal growth factor; lactoperoxidase; and lactoferrin. Depending on the health of the cows, feeding practices followed, collection period, and the processing/concentration practices utilized by specific manufacturers, the actual range and quantities of specific macro- and micronutrients, Ig, cytokines, growth factors, and other compounds might vary considerably.37,38,39 BC contain relatively high amounts of Ig. Typically, Ig in general, and IgG specifically, constitutes the largest contribution to protein content in BC, with lactalbumin and casein contributing lesser amounts. Mean percentages of fat, protein, and lactose in colostrum were 6.7, 14.9, and 2.5, respectively.40 Other reports have estimated Ig concentrations from bovine mammary secretions with ranges for IgG1 (52-87 g/L), IgG2 (1.6-2.1 g/L), IgM(3.7-6.1 g/L), and IgA (3.2-6.2 g/L).41,42 As a rule, the Ig contribution will decline substantially in any BC collected more than 24 hours post-parturition and the amount of lactalbumin and casein will increase proportionately.

1. Detailed research plan (Materials and Methods) 5.1) Study design and setting This will be Double blind Randomized Placebo controlled Trial. The sample size was taken from Singh et al which compared Granulocyte Colony-Stimulating factor (G-CSF) versus Standard Medical Treatment (SMT) in patients with sAH [MELD score was 27.5 (IQR: 19 - 41) and the DF score was 77.4 (IQR: 37-235)]. This was the only available Indian study with similar baseline MELD and mDF scores before starting our study. Eighteen out of twenty three (78.26%) in Group A (G-CSF + SMT) survived at 90 days vs. 5/23 (21.74%) in Group B (SMT). Our study randomized patients with comparable baseline disease severity - Median MELD and DF scores were 30.67 (IQR: 27.87-33.39) and 78.42 (IQR: 57.79- 95.50), respectively The Singh et al (Singh V, Sharma AK, Narasimhan RL, Bhalla A, et al Granulocyte colony-stimulating factor in severe alcoholic hepatitis: a randomized pilot study. Am J Gastroenterol. 2014 Sep;109(9):1417-23) study had a poor survival rate in the Placebo arm (22%), but the effect size we used in our study was modest and was intentionally chosen to be substantially smaller than that observed in the Singh et al. trial, reflecting the exploratory nature of Colostrum therapy. Assuming a survival improvement from 22% to 42%, the required sample size was estimated to be 78 participants per group. Accounting for a 10% dropout rate, the final sample size was set at 87 participants per arm, resulting in a total of 174 participants. [STATA version 16 (Stata Corp, College Station, TX, USA)] The study will be conducted on patients of Severe Alcoholic Hepatitis admitted in

• Principal Investigator: Sandeep S Sidhu, Dayanand Medical College and Hospital, Ludhiana, Punjab, India
• Principal Investigator: Ajay Dusseja, PGIMER, Chandigarh New
• Principal Investigator: Shalimar, AIIMS, Delhi, India
• Principal Investigator: Sandeep Nijhawan, SMS Hospital, Jaipur, India
• Principal Investigator: Dharmesh Kapoor, Global Hospital, Hyderabad, India Informed consent shall be taken from the patient/nearest relative of the patient for enrollment in the trial.

Assignment Participants were randomly assigned (1:1) to BC or placebo via a secure, centralized web-based randomization system (Trans European Network for Clinical Trials Services - ALEA, Netherlands) with block randomization stratified by study center. Allocation concealment was maintained throughout the study. Participants, investigators, study coordinators, treating physicians, outcome assessors, and statisticians remained blinded until the final analyses were completed.

Masking Participants, investigators, study coordinators, treating physicians, outcome assessors, and statisticians remained blinded until the final analyses were completed.Each center will have serially numbered identical satchets containing bovine colostrum or pasteurized bovine milk administered by a personnel according to the random sequence. The drug and the placebo will be identical in color, taste, appearance to ensure masking. All study personnel will be blinded to the treatment assignment (Bovine colostrum and placebo) for the duration of the study. The study drug will be administered by an oral route. The randomization code will be revealed only after the completion of recruitment, data collection and data analysis has been carried out.

6.3.3 Randomization implementation Enrolment of patients, assessing eligibility and obtaining informed consent will be carried out by Principal Investigator (S.S. Sidhu)and other Centers Principal Investigator.

6.3.4 Study Intervention The patients will be randomized to either Experimental arm: Bovine colostrum Enteral nutrition: Protein 1.5 gm/kg/day, energy (kcal) 30-40/day, B complex vitamins daily.

Pasteurized Bovine colostrum as a freeze dried powder (20 gm thrice a day) for 4 weeks.

+ Antibiotics + Diuretics +Terlipressin for HRS + acid suppression for prophylaxis against gastrointestinal hemorrhage + EVL for variceal bleed +drugs for HE if indicated

Placebo Comparator: Placebo Enteral nutrition: Protein 1.5 gm/kg/day, energy (kcal) 30-40/day, B complex vitamins daily.

Placebo (Pasteurised Milk Powder) 20 gms thrice a day for 4 weeks[50% protein enriched with whey protein isolates (0.05% to 0.1% IgG), 20% fat, 25% carbohydrates, and 480 kcal/100 g]

• Antibiotics + Diuretics + drugs for HE + Terlipressin for HRS + acid suppression for prophylaxis against gastrointestinal hemorrhage + EVL for variceal bleed + if indicated.

1. 5.2. Inclusion Criteria: *Inclusion criteria:
2. Onset of jaundice within the prior 8 weeks
3. Ongoing consumption of > 40 (female)g or > 60 (males) g alcohol/day for 6 months or more, with less than 60 days of abstinence before the onset of jaundice
4. Aspartate aminotransferase (AST) > 50, AST/alanine aminotransferase (ALT) >1.5, and both values < 400 IU/L
5. Serum bilirubin (total) > 3.0 mg/dL
6. Maddrey's discriminant function (DF) > 32
7. Model for End-stage Liver Disease (MELD) score > 20 *Exclusion Criteria: <!-- -->
8. Uncontrolled infections
9. Multiorgan failure
10. Uncontrolled upper gastrointestinal bleeding#
11. Preexisting kidney injury with serum creatinine > 2.5 mg/dL
12. Other chronic liver diseases
13. Hepatocellular carcinoma, other active malignancies, except skin cancer
14. Pregnancy
15. Uncontrolled substance use disorder
16. Known cow milk allergy or severe lactose intolerance**
17. Treatment with Corticosteroids *Crabb DW, Bataller R, Chalasani NP, et al. Standard Definitions and Common Data Elements for Clinical Trials in Patients With Alcoholic Hepatitis: Recommendation From the NIAAA Alcoholic Hepatitis Consortia. Gastroenterology 2016;150(4):785-790. #Patients with upper Gastrointestinal bleeding controlled with vasopressors and endoscopic haemostasis, were included in the study. ** The diagnosis of Lactose Intolerance was made clinically and confirmed by the Hydrogen Breath Test. 5.3) Study Methods Enrolment of patients, assessing eligibility and obtaining informed consent will be carried out by P.I. of each center 5.3.1.) Study Intervention Treatment for SAH: <!-- -->
18. Enteral nutrition: Protein 1.5 gm/kg/day, energy (kcal) 30-40/day, B complex vitamins daily.
19. Pasteurized Bovine colostrum (20gmthricea day) for 4 weeks in one arm or Placebo (Pasteurized milk powder)20gms thrice a day for4 weeks . 5.4.0) Laboratory tests Liver biopsy: Transjugular liver biopsies will be obtained before start of therapy diagnosis of Alcoholic hepatitis [if possible] 5.4.1) Biochemical tests Hemogram, blood glucose, liver function tests, prothrombin time, serum electrolytes, blood urea and serum creatinine will be done at baseline, and at and 7th day,14 days, 21 days , 30 days and then bimonthly for two monthsor earlier if indicated. 5.4.2) Microbiologic tests: A diagnostic paracentesis will be done in all patients with ascites, at baseline, to diagnose Spontaneous Bacterial Peritonitis. A repeat cell count (Total and differential) shall be done on day 5 in patients diagnosed to have Spontaneous Bacterial Peritonitis. Blood culture, urine culture, cultures of aspirates from endotracheal tubes in ventilated patients for aerobic and anaerobic bacteria, and fungi shall be done on at admission. A chest radiograph shall also be done. Endotoxin level estimation, alpha TNF,IL6\& IL8 will be done at baseline and at end of treatment. 5.4.3) Tests for etiologic evaluation: Etiology will be taken as alcohol if there is a history of significant alcohol intake (20 gm/day for females, and 80 gm/day for males for 10 years or more). Each patient will be tested for Hepatitis B surface antigen (HBsAg) and Anti-hepatitis C (HCV) antibody using a third generation commercial ELISA. Wherever indicated, autoimmune hepatitis will be diagnosed using antinuclear antibody, anti-smooth muscle antibody and anti-LKM; and hemochromatosis using serum iron, TIBC, ferritin and transferrin saturation. Each patient shall have an abdominal ultrasonography. Upper gastrointestinal endoscopy would be done, if indicated. Ascitic Fluid analysis:Ascitic Fluid analysis will be done on 1st day and 5th day of treatment. 5.5) Outcome measures and endpoints 5.5.1) Primary outcome measure Survival at 3 month 5.5.2) Secondary outcome measure a. Survival at 1 month b. Change in mDF levels/ MELD will be measured at baseline and after 30 days of treatment c. Change in Endotoxin levels will be measured at baseline and after 30 days of treatment d. Change in Cytokines levels will be measured at baseline and after 30 days of treatment e. Number of episodes of infection (bacteremia, Pneumonia, SBP, Cellulitis, UTI) 5.5.3) Study Endpoints 1.Liver related death 2. Liver Transplantation 3. Death due to other causes 4. Complication of Disease (GI Bleed, Renal failure, Sepsis) 5.6) Monitoring for Adverse Events: Any adverse event will be recorded specifying the time of onset, the duration, the severity and the relationship to the test medication. 5.7) Tolerability Tolerability of the study drug will be assessed by comparison of full blood cell count, liver function tests and renal function tests at baseline and at end of treatment. 5.8) Follow-up All patients will be followed up to 3 months or until death. The status alive or dead will be assessed by telephoning a family member or by contacting the death registry at patients at birth place or place of residence. 5.9) Statistical Analysis: The primary efficacy analysis will adhere to the intention-to-treat principle. Overall survival will be estimated using the Kaplan-Meier method and compared using the log-rank test. Hazard ratios (HRs) and 95% confidence intervals (CIs) will be estimated using Cox proportional hazards regression. A Priori Adjustment: We have pre-specified key known prognostic confounders such as treatment group, Site /Center, Grade of Ascites, Acute Kidney Injury, Age (years) , Alcohol Intake(gms), MELD score in our statistical analysis plan and they will be adjusted in the Cox proportional hazards regression model. The proportional hazards (PH) assumption was assessed using scaled Schoenfeld residual plots. A p-value of Schonfeld < 0.05 indicates that the null hypothesis ~ hazard ratio is constant over time, is rejected i.e. the proportional hazards assumption gets violated. When the Proportional Hazards (PH) assumption is violated, the hazard ratio changes continuously over time. Reporting a single, average hazard ratio from a standard Cox model becomes mathematically flawed and clinically misleading. To address a violation of the PH assumption, the restricted mean survival time (RMST) for both treatment groups will be analyzed over 90 days. Restricted Mean Survival Time (RMST) resolves this issue through three distinct mechanisms: <!-- -->
20. It Drops the Proportionality Hazard Assumption Entirely The standard Cox model relies on the assumption that the relative risk between treatment groups is identical at days 1, 100, and 1000.

• RMST makes no assumptions about the shape or parallelism of the survival curves.
• It evaluates the mathematical area under the Kaplan-Meier survival curve from time zero up to a specific time horizon ~ tau. Because it evaluates the overall space under the curve rather than the shifting distance between lines at given points, the calculation remains robust and valid even if the survival curves cross, diverge, or plateau.

1. It Provides an Absolute Summary Metric

• By analyzing the RMST for both groups, you calculate the average event-free survival for a patient within that specific window of time.
• Because it integrates the entire survival experience over the window, it naturally factors in the period where the treatment performed worse alongside the period where the treatment performed better, balancing out the time-varying effects.

All this well captured by an editorial by Han L. Breaking Free from the Hazard Ratio: Embracing the Restricted Mean Survival Time in Clinical Trials. NEJM Evid. 2023 Aug;2(8):EVIDe2300142. doi: 10.1056/EVIDe2300142. Epub 2023 Jul 25. PMID: 38320152.

Continuous secondary outcomes were analysed using normality tests (Quantile-Quantile plots and Kolmogorov-Smirnov tests) and were compared between groups using appropriate parametric or non-parametric tests.

Joint longitudinal-survival analysis of MELD and mDF scores To account for the association between longitudinal continuous measurements of MELD / mDF scores and time-to-event outcomes, a joint modelling approach was applied. The time variable in the R Model was coded in 0,1,2,3,4 weeks. Joint modelling was performed by simultaneously fitting two linked submodels to evaluate the relationship between longitudinal MELD/mDF scores and time-to-event outcomes. The longitudinal submodel was specified as a linear mixed-effects model to describe changes in MELD and mDF scores over time. This model was used to assess whether MELD/mDF scores increased or decreased during follow-up and to compare score trajectories between the BC and Placebo groups, while accounting for within-subject correlation due to repeated measurements. The survival submodel was used to analyse time-to-event outcomes comparing BC versus Placebo. Within the joint modelling framework, the survival process was specified using a Weibull accelerated failure time formulation. The two submodels were linked through a time-dependent association structure, allowing the longitudinal MELD/mDF score trajectories to directly influence the risk of the event. Joint models were fitted using the JM package in R (1).

Recurrent episodes of infections were analyzed using generalized estimating equations (baseline infections) and partial proportional odds models (infections occurring during the treatment period).

The sample size provided 80% power to detect a 20% absolute improvement in 90-day survival at a two-sided α level of 0.05.

We are planning a study of 2 independent groups in the ratio of 1:1. Prior dataindicates that when 40 patients undergoing gastrointestinal surgery were randomized and given 56 g daily of Bovine Colostrum for 3 days preoperatively) or placebo and the course of the plasma endotoxin levels and the endotoxin neutralization capacity (ENC) were measured daily up to the 10th postoperative day 10. The results showed that the LPS-levels in the LC1-group, expressed as AUC, were significantly lower than those in the control group (p < 0.05). The difference between the two groups was apparent on the day of the operation and the day after. The fall in endotoxin neutralizing capacity (ENC) was significantly lower in the patients treated with the BCC and the return to initial values was faster than in the control group (p < 0.006).

In the second randomized study, 40 gms of Bovine Colostrum were given perioperatively to 60 open heart surgery patients without any decrease in LPS levels or fall in the ENC.11