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Immunopharmacology and Immunotoxicology, 28:1–7, 2006Copyright Informa HealthcareISSN: 0892-3973 print / 1532-2513 onlineDOI: 10.1080/08923970601067045 g , Vol. 28, No. 4, November 2006: pp. 1–14 A Pilot Study of an anti-MRSA
Bio-Engineered Lacteal
Complex (anti-MRSA BLC) in a
Murine Septicemia Model

Nix, and D.W. DeYoung Jesse A. Stoff,1 David E. Nix,2 and D.W. DeYoung3 1Tiburon Laboratory, Tucson, Arizona, USA 2Department of Pharmacy Practice & Science, University of Arizona College ofPharmacy, Tucson, Arizona, USA 3University Animal Care, University of Arizona, Tucson, Arizona, USA Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen of 10humans and other animals, causing septicemia, abcessation, toxemia, and other infec-tious diseases. Refined bioengineered lacteal complex (BLC), made specifically againstMRSA, is a novel complex of low molecular weight immunogenic and antimicrobialmolecules. It was evaluated in vivo using a mouse model of MRSA-induced peritonitis.
Intraperitoneal dosing of anti-MRSA BLC demonstrated a therapeutic effect (83% 15survival) against an intraperitoneal MRSA challenge that caused 100% mortality inuntreated animals. Anti-MRSA BLC is a promising therapeutic modality for MRSAinfection.
Bio-engineered Lacteal Complex (BLC), Methicillin-Resistant Staphylo-coccus Aureus (MRSA), Murine Peritonitis, Sepsis, Toxemia.
Methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly com-mon cause of life- and limb-threatening infections throughout the world.(1–3) Itis now one of the most common causes of nosocomial and community-acquiredinfections and has developed a stable resistance to many antibiotics.(4) Since 251961, when MRSA was first reported as a clinical concern, it has become a Dr. Stoff is the lead inventor of BLC and does not have any financial interest inBerlett, Inc. Berlett, Inc. does not have any financial interest in BLC. Dr. DeYoung andDr. Nix have nothing to declare.
Address correspondence to Dr. Jesse A. Stoff, Tiburon Laboratory, 2661 N. Camino DeOeste, Tucson, AZ 85745, USA; E-mail: thedoc@drstoff.com J.A. Stoff, D.E. Nix, and D.W. DeYoung serious problem with increasing numbers of cases and associated morbidityand mortality.(5,6) A filtrate of colostrum and whey from antigen infused dairy cows has been used experimentally since the late 1950s in humans and animals.(7) Empiricaland anecdotal evidence suggests enhanced immune-boosting properties com- 35pared with typical colostrum and whey products. From the beginning, thismaterial appeared to restore immune function in people and animals sufferingfrom conditions related to immune dysfunction including those suffering fromacute, chronic, and degenerative diseases. Further research and developmenthas yielded a novel (U.S. Patent Application No. 11/122,231) bioengineered 40lacteal complex (BLC) that utilizes new technology for its induction, purifica-tion, and characterization of molecular components. Anti-MRSA BLC containsspecific, low molecular weight molecules that have antimicrobial propertiesagainst MRSA and also enhances immune function against MRSA infection.
The present study was undertaken to investigate in vivo effects of anti- 45 MRSA BLC using an intraperitoneal systemic sepsis model.
MATERIALS AND METHODS
A clinical isolate of a community acquired MRSA was utilized. Susceptibil- ity testing using the Vitek® instrument at a certified clinical laboratory against 5017 antibacterial agents showed susceptibility to gentamicin, nitrofurantoin,rifampin, vancomycin, tetracycline, and trimethoprim/ sulfamethoxazole.
Bacterial suspensions of MRSA (in log growth phase) in 3% Todd Hewitt media (BD, BBL ref # 211736, Sparks, MD, USA) were freshly prepared froman overnight culture on 5% sheep blood agar (Remel Blood Agar, ref 01202, 55Lenexa, KS, USA). The optical density was adjusted to 8% transmittanceusing a Vitek® colorimeter and this correlated with a 1 × 1010 per mL bacte-rial count. The inoculum concentration was confirmed by quantitative serialdilution with the spread plate method.
The anti-MRSA BLC material used in the study was specifically manufac- tured for that purpose making use of a new (patent applied for) technology.
Chemically free raised cows went through immune induction using MRSA cellfragments along with cytokine adjuvants, inoculated into the wall of the udderwith a CO2 powered needleless "gun." After gathering milk from the cows, the 65milk was processed to remove all material with a molecular weight greaterthan 100 kD. The 100 kD filtrate was then evaluated for antimicrobial effectagainst MRSA, with growth inhibition curves on SRBC plates, as one Anti-MRSA BLC bio-logical marker for immune induction. After purifying the material it wasstandardized to 8 mg/mL of solids and assayed for its content of key molecules 70including defensins, granulysins, and specific transfer factors.
Young (10 week old, 24+/−2 g) BALB/c mice were used in this study. The study was approved by the University of Arizona Institutional Animal Careand Use Committee and the Institutional Bio-Safety Committee. Animal 75experiments were conducted at the University of Arizona Animal Care Facil-ity and procedures adhered to all applicable standards for animal care. Themice were kept 3 to a cage and all the animals had unrestricted access to foodand water throughout the study. They were kept in a BL 3 facility at standardtemperatures and humidity. Each MRSA-dose test group consisted of 3 male 80and 3 female mice in order to establish the concentration of MRSA needed toinduce terminal morbidity. The anti-MRSA BLC therapeutic group consistedof 12 male and 12 female mice. Animals demonstrating terminal morbidity(only minimally responsive to stimuli, labored breathing, and severedehydration) were euthanized. Since BLC is a known immunogen, which is an 85important part of its mechanism of action, mice with normal immune systemswere used.
In Vitro Experiments
Electrophoresis was used to evaluate the presence of peptides and molecules below 100 kD in the study drug materials. The presence of specific 90small molecular peptides is known to modulate the immune system. Over100 different molecules were found to be present which constituted the 12major bands demonstrated in the 18 % tris-glycine electrophoresis gel. Manyof the molecules were identified by mass spectroscopy, amino acid sequenc-ing, and other technologies. These molecules included cytokines, mini- cytokines, defensins, granulysins, transfer factors, antibody fractions, andlactoferrin.
Flow cytometry experiments were done on whole blood to calculate a theo- retical maximal safe dose of the BLC based upon its immunogenic effects. Forthese experiments increasing amounts of BLC were added to 100 μL aliquots 100of whole blood, and at hourly intervals the blood was analyzed by flow cytome-try for lymphocyte, monocyte, and neutrophil cell activation by measuringCD69 levels and cell movement. The activation curves were plotted and theoptimal dose was calculated. Once the concentration of BLC exceeded 10% ofthe total volume there was less activation and an increase in cellular apopto- 105sis was noted.
The dose utilized for the mouse model was determined to be 10% of the estimated blood volume (0.13 mL). Groups of 3 male and 3 female mice were J.A. Stoff, D.E. Nix, and D.W. DeYoung administered intraperitoneal doses of 0.13 mL anti-MRSA BLC for acuteassessment. This group was closely observed for 2 weeks for their level of 110activity, responsiveness, feeding, and changes in body weight and showed nodeviation from the expected normal behaviors.
Mouse Peritonitis Sepsis Model
The model chosen for this study was that of intraperitoneal systemic sepsis. This model has been well described and is a standard method of testing 115antimicrobial agents.(8,9) The lower abdomens of the mice were swabbed withalcohol and briefly allowed to dry. The mice were then inoculated in their leftlower quadrant, intraperitoneally with of 0.1 mL of MRSA inoculum. Prelimi-nary studies demonstrated that a 1 × 1010 cfu/mL suspension of MRSA(infective dose of 1 × 109 bacteria) induced 100% mortality, and this was used 120for the control and therapeutic groups included in this study. The controlgroup, which received intraperitoneal MRSA, was observed every 2 hr andtheir apparent health recorded. The test group received the same inoculum ofMRSA but they also received anti-MRSA BLC.
The test group was given an intraperitoneal injection of 0.13 mL anti- 125 MRSA BLC, immediately after the bacterial challenge. Based upon in vitrostudies, the effect of the BLC wanes by 4 hr. For mice in the test group thatbecame significantly incapacitated, further doses of anti-MRSA BLC weregiven at 4-hr intervals. The mice were given no more than three doses ofanti-MRSA BLC per day. At the end of the study the mice were humanely 130euthanized.
Measurement of Impairment
The mice were observed three times per day and their apparent health was recorded. The endpoint of the study was either death or "severely mori-bund" at which point the mice were sacrificed. However, data on their relative 135levels of impairment were recorded as part of the observational report.
"Impaired" mice were defined as having diarrhea and mild dehydration. Theystill reacted normally to stimuli and continued to eat and drink normally.
These mice could receive further doses of anti-MRSA BLC up to three dosesper day. Moribund mice had severe diarrhea and/or severe dehydration. They 140reacted sluggishly or not at all to stimuli, had labored breathing, and did nottry to eat or drink.
Statistical analysis was accomplished using SAS version 8.2 (SAS Inc., Cary, NC, USA). Fisher's exact test was used to compare frequency 145data.
Anti-MRSA BLC Figure 1 demonstrates the rapid induction of terminal morbidity thatoccurred in the untreated mice following intraperitoneal inoculation with theMRSA. They developed severe diarrhea very quickly followed by dehydration. 150They then became lethargic, obtunded, and all 6 died within 12 hr. Severalgroups of mice were required to establish the dose of MRSA needed. Anintraperitoneal injection of 0.1 cc of 1 × 1010 cfu/mL caused 100% terminalmorbidity.
Among anti-MRSA BLC treated mice, 20/24 (83%) survived compared 155 with 0/6 (0%) of untreated mice (p < 0.001). After an initial 48 hr, post-MRSAinoculation, none of the surviving mice showed any lingering signs of illness ordehydration. They were observed to feed and drink normally, respond nor-mally to stimuli, and their skin turgor returned to normal. After the 8:00 a.m.
observation on the 8th day, mice were all humanely euthanized.
All female mice survived compared with 8/12 (67%) of male mice. Given the small sample size this difference in survival by sex was not statisticallysignificant (p=0.093) Table 1 shows the morbidity response of the male micegiven an inoculum of MRSA and then treated with anti-MRSA BLC. Two malemice were more significantly impaired and required reinjection with the anti- 165MRSA BLC of which 1 survived. The survival rate of the female mice was100% (12 of 12 mice). One female mouse was more significantly impaired andrequired reinjection with the anti-MRSA BLC followed by a full recovery.
MRSA represents a common cause of nosocomial infections and in the past 170decade has emerged as an important cause of community acquired infections.
The frequency of methicillin-resistance among isolates of S. aureus ecim 5 gniv4ivru3s fo r2ebm1uN0
Time in hours post inoculation with MRSA
Figure 1: Number of surviving control (nontreated) mice postinoculation with MRSA.
J.A. Stoff, D.E. Nix, and D.W. DeYoung Table 1: Morbidity response of the male mice given an inoculum of MRSA and then
treated with anti-MRSA BLC.
Study day
All surviving mice (n = 8) were considered "normal" on days 4–8.
approximates 50% of hospital isolates and 15% of community acquired isolates.
MRSA infections have been associated with greater health care expendituresand increased mortality compared with methicillin-susceptible S. aureus. 175Although vancomycin is considered the treatment of choice for serious infec-tions involving MRSA, issues relating to therapeutic failures, poor penetrationin certain body sites, and vancomycin resistance are major concerns. Recentintroductions of the oxazolidinone, linezolid, and the lipopeptide, daptomycin,have provided new options; however, the role of these agents is often debated. 180Many problems with therapeutic management of MRSA infections remain.
BLC represents a new therapeutic direction by inducing an immunological response against the microbe for which it was made. An immune counter-attack by the host may limit the impact of resistance and may prove to be animportant addition to the therapeutic armamentarium in the future.
In this study, females tended to respond better to anti-MRSA BLC therapy than male mice. Males appear more susceptible to MRSA infection,perhaps suggesting hormonal involvement in host response.(10) Sex hormonesmay influence bacterial growth and survival directly.(11) Estrogen is known toaccelerate inflammation and the immune response in both mice and 190humans.(12) It has been postulated that estrogen has a differential effect on Tand B cell mediated immune responses. Although survival differences by sexwere not statistically significant (p = 0.093) in this study, future studies needto address this potential sex difference in response. If sex differences inresponse are confirmed, future studies will be required to ascertain whether a 195different dose or administration schedule of anti-MRSA BLC will overcomethe difference.
This investigation was supported, in part, by a research grant from Berlett,Inc.
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5. Jevons, M.P.; Coe, A.W.; Parker, M.T. "Celbenin" resistant staphylococci. Br. Med. 210 J. 1961, 1, 124–125.
6. Hrynewicz, W. Epidemiology of MRSA. Infect. (suppl. 2): 1999, S13–S16.
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isolates. Can. J. Microbiol. 2000, 46, 920–926.
11. Warren, G.L.; Gray, J.; Vernon, S.K. The in vitro action of mammalian sex 225 hormones on Staphylococcus aureus. Chemotherapy (Basel) 1970, 15, 273–285.
12. Carlsten, H.; Nilsson, N.; Jonsson, R. et al. Differential effects of oestrogen in murine lupus: acceleration of glomerulonephritis and amelioration of T cell
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Organic Anion Transporter 3 Contributes to theRegulation of Blood Pressure Volker Vallon,*†‡ Satish A. Eraly,* William R. Wikoff,§ Timo Rieg,*‡ Gregory Kaler,*David M. Truong,* Sun-Young Ahn,* Nitish R. Mahapatra,* Sushil K. Mahata,*‡Jon A. Gangoiti,储 Wei Wu,* Bruce A. Barshop,储 Gary Siuzdak,§ and Sanjay K. Nigam*储¶ Departments of *Medicine, †Pharmacology, 储Pediatrics, and ¶Cellular and Molecular Medicine, University ofCalifornia, San Diego, ‡Department of Medicine, San Diego VA Healthcare System, and §Department of MolecularBiology and the Center for Mass Spectrometry, Scripps Research Institute, La Jolla, California

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