Lymphatic Pump Treatment as an Adjunct to Antibiotics for Pneumonia in a Rat Model

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1 Editor s Note: Corrections to this rticle were pulished in the June 2015 issue of The Journl of the Americn Osteopthic Assocition (2015;115[6]:357). The corrections hve een incorported in this online version of the rticle, which ws posted June An explntion of these chnges is ville t Lymphtic Pump Tretment s n Adjunct to Antiiotics for Pneumoni in Rt Model Lis M. Hodge, PhD; Citlin Cresy, MS; KihRe Crter, MS; Ashley Orlowski, BS; Artur Schnder, DO, PhD; nd Hollis H. King, DO, PhD From the Deprtment of Cell Biology nd Immunology (Dr Hodge, Ms Crter, nd Ms Orlowski) nd The Osteopthic Reserch Center (Dr Hodge) t the University of North Texs Helth Science Center in Fort Worth; the Deprtment of Melnom Medicl Oncology t the University of Texs MD Anderson Cncer Center in Houston (Ms Cresy); the Deprtment of Grdute Medicl Eduction t Peconic By Medicl Center in Riverhed, New York (Dr Schnder); nd the Deprtment of Fmily nd Prevention Medicine t the University of Cliforni, Sn Diego (Dr King). Finncil Disclosures: None reported. Support: Funding for this study ws provided y the Ntionl Institutes of Helth (grnt AT004361) nd the Americn Osteopthic Assocition (grnts nd ). Address correspondence to Lis M. Hodge, PhD, University of North Texs Helth Science Center, 3500 Cmp Bowie Blvd, Fort Worth, TX E-mil: lis.hodge@unthsc.edu Sumitted Septemer 16, 2014; revision received Decemer 23, 2014; ccepted Jnury 28, Bckground: Lymphtic pump tretment (LPT) is technique used y osteopthic physicins s n djunct to ntiiotics for ptients with respirtory trct infections, nd previous studies hve demonstrted tht LPT reduces cteril lod in the lungs of rts with pneumoni. Currently, it is unknown whether LPT ffects drug efficcy. Ojective: To determine whether the comintion of ntiiotics nd LPT would reduce cteril lod in the lungs of rts with cute pneumoni. Methods: Rts were infected intrnslly with colony-forming units (CFU) of Streptococcus pneumonie. At 24, 48, nd 72 hours fter infection, the rts received no therpy (control), 4 minutes of shm therpy, or 4 minutes of LPT, followed y sucutneous injection of 40 mg/kg of levofloxcin or sterile phosphte-uffered sline. At 48, 72, nd 96 hours fter infection, the spleens nd lungs were collected, nd S pneumonie CFU were enumerted. Blood ws nlyzed for complete lood cell count nd leukocyte differentil count. Results: At 48 nd 72 hours fter infection, no sttisticlly significnt differences in pulmonry CFU were found etween control, shm therpy, or LPT when phosphte-uffered sline ws dministered; however, the reduction in CFU ws sttisticlly significnt in ll rts given levofloxcin. The comintion of shm therpy nd levofloxcin decresed cteril lod t 72 nd 96 hours fter infection, nd LPT nd levofloxcin significntly reduced CFU compred with shm therpy nd levofloxcin t oth time points (P<.05). Colony-forming units were not detected in the spleens t ny time. No sttisticlly significnt differences in hemtologic findings etween ny tretment groups were found t ny time point mesured. Conclusion: The results suggest tht 3 pplictions of LPT induces n dditionl protective mechnism when comined with levofloxcin nd support its use s n djunctive therpy for the mngement of pneumoni; however, the mechnism responsile for this protection is uncler. J Am Osteopth Assoc. 2015;115(5): [Pulished correction ppers in J Am Osteopth Assoc. 2015;115(6):357.] doi: /jo Community-cquired pneumoni (CAP) ccounts for more thn 1 million hospitl dmissions ech yer worldwide. 1 Notwithstnding the costs of ntiiotics for the mngement of CAP, hospitl room nd ord is the single most expensive component of helth cre in the United Sttes. The men length of hospitl sty for CAP is 5.6 dys for ptients ged 65 yers or older nd 4.5 dys for younger ptients, 2 mking the cost of hospitliztion for CAP more thn $8 illion nnully. 3 Shortening the length of hospitl sty y 1 dy could sve more thn $1 illion per yer; therefore, 306 Downloded from y guest on 01/15/2019 The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No. 5

2 therpies tht shorten hospitliztion or llow outptient mngement should e exploited. Lymphtic pump tretment (LPT) comprises osteopthic mnipultive medicine techniques tht trget the musculoskeletl system nd enhnce the flow of lymph through the lymphtic system. 4,5 Cliniclly, LPT is reported to increse vccine-specific ntiodies, 6,7 reduce the need for intrvenous ntiiotics, 8,9 protect ginst lower respirtory trct disese, 8-10 nd shorten the durtion of hospitl sty in elderly ptients with pneumoni. 9 To our knowledge, no reports hve een pulished mesuring the effects of LPT on the lymphtic system in humns, ut niml studies hve demonstrted tht LPT yields significnt (P<.05) increses in lymph flow nd in the lymphtic concentrtion of leukocytes In these studies, LPT did not preferentilly moilize ny specific immune cell popultion, ut it significntly (P<.05) incresed thorcic duct lymph flow nd totl leukocyte concentrtions, resulting in n pproximtely 10-fold increse in the lymphtic flux. 12 Lymphtic pump tretment lso significntly (P<.05) enhnced the lymphtic flux of inflmmtory cytokines, chemokines, nd rective oxygen nd nitrogen species in oth thorcic nd mesenteric lymph, 17,18 nd it enhnced the uptke of protein from the tissue s interstitil spce. 19 Collectively, these studies suggest tht LPT cn enhnce the lymphtic nd immune system responses, which my ccelerte the clernce of pneumococcl cteri. A study pulished in used rt model to investigte the effect of LPT on lymphtic function. Rts received LPT in mnner similr to tht reported previously, 19 nd lymph ws collected from the cistern chyli. Four minutes of LPT cused sttisticlly significnt increse in lymph flow nd leukocyte concentrtions. These results were consistent with findings in dogs 11-13,16 nd demonstrted the enhnced lymph flow nd the lymphtic relese of immune cells induced y LPT in smller niml. In lter study, 14 rts were infected intrnslly with Streptococcus pneumonie nd received shm therpy or LPT. The ppliction of LPT once dily for 7 consecutive dys reduced the concentrtion of cteri in the lungs pproximtely 30-fold compred with shm therpy. In follow-up study, Cresy et l 20 found tht 3 pplictions once dily for 3 consecutive dys of either thorcic or dominl LPT were le to significntly (P<.05) reduce the numers of pulmonry S pneumonie colony-forming units (CFU) compred with control or shm therpy. These results suggest tht LPT my protect ginst cteril pneumoni y inhiiting cteril growth in the lung; however, the mechnism responsile for this clernce is still under investigtion. To our knowledge, no study hs een pulished to show tht lymphtic tretments, such s LPT, ffect drug efficcy. In the current study, we hypothesized tht the comintion of ntiiotics nd LPT would reduce the concentrtion of cteri in the lungs of rts infected with S pneumonie. Antiiotics re generlly used to mnge cteril pneumoni nd other infectious diseses; therefore, it is importnt to identify how complementry therpies, such s LPT, ffect ntiiotic efficcy. Methods Animls The present study ws pproved y the Institutionl Animl Cre nd Use Committee nd conducted in ccordnce with the Guide for the Cre nd Use of Lortory Animls. 21 Mle inred Fischer 344 rts, free of cliniclly evident signs of disese nd weighing etween 200 to 300 g, were used. The rts hd indwelling jugulr vein ctheters, which remined in plce for the durtion of the study. Infection Anesthesi ws delivered (30 mg/kg ketmine nd 5 mg/ kg xylzine), nd the rts were intrnslly inoculted with S pneumonie (ATCC 6301) CFU in 100 µl of phosphte-uffered sline (PBS). After infection, the rts were held verticlly for few seconds to llow for spirtion of the fluid. No sustntil weight loss or ny The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No Downloded from y guest on 01/15/2019

3 other clinicl signs of disese were oserved in the nimls during the study (dt not shown). Therefore, disese free ws defined s n sence of S pneumonie in lung homogentes. resuspended in sterile PBS. At 24, 48, nd 72 hours fter infection, rts received no mnul therpy (control), shm therpy, or LPT followed y sucutneous injection of 40 mg/kg of levofloxcin or sterile PBS. Leukocyte Enumertion Blood smples were collected y mens of crdic puncture nd drwn into EDTA-coted vcutiner lood test tues. Totl leukocytes nd differentil leukocyte count were enumerted using n utomtic hemtology nlyzer. Bcteril Enumertion S pneumonie ATCC 6301 stocks were stored t 80 C in rin hert infusion roth contining 10% glycerol until use. Bcteri were cultured on trypticse soy gr with 5% sheep lood gr pltes nd incuted overnight t 37 C nd 5% cron dioxide. The cteri were collected y wshing the pltes twice with sterile PBS nd diluted to n opticl density t 600 nm for infection. The CFU concentrtion in the suspension ws determined retrospectively y seril dilution nd plting on trypticse soy gr with 5% sheep lood gr pltes. For enumertion of cteri in pulmonry nd spleen tissue, lungs nd spleens were removed nd homogenized seprtely for 25 seconds using tissue homogenizer. Ten-fold (1:10 to 1:1,000,000) seril dilutions were mde in 96-well plte. Twenty microliters of ech dilution were plted onto trypticse soy gr with 5% sheep lood pltes in duplictes. The pltes were incuted overnight t 37 C with 5% cron dioxide, nd CFU were counted fter 18 hours. Levofloxcin The sensitivity of the ATCC 6301 strin of S pneumonie to levofloxcin ws confirmed in vitro using the Kiry Buer susceptiility test protocol (dt not shown). Dosge studies were performed to determine the optimum concentrtion of levofloxcin to e used in vivo. Rts were intr nslly infected with CFU nd received sterile PBS or 25, 40, or 50 mg/kg of levofloxcin Interventions Adominl LPT ws pplied to rts s previously descried. 14,15,20 Briefly, nesthetized (10 mg/kg propofol) rts were kept in right lterl recument position. To perform LPT, the opertor (A.S.) contcted the domen of the rt with the thum on 1 side nd index finger nd middle finger on the other side of the medil sgittl plne. The fingers were plced ilterlly cudd to the ris. Sufficient pressure ws exerted medilly nd crnilly to compress the lower ris until sustntil resistnce ws met ginst the diphrgm, then the pressure ws relesed. Compressions were dministered t rte of pproximtely 1 per second for the durtion of the 4 minutes of tretment. During shm therpy, rts were nesthetized nd the opertor contcted the domen of the rt for 4 minutes in mnner similr to LPT; however, no compressions were pplied. This procedure ws designed y H.H.K. to simulte the LPT used in humns. 15 Dt Collection At 48, 72, nd 96 hours fter infection, the rts were euthnized in ccordnce with the Americn Veterinry Medicl Assocition guidelines. The lungs nd spleens were collected nd the concentrtions of S pneumonie were mesured in ech tissue. Blood smples were nlyzed t 0, 24, 48, 72, nd 96 hours for complete lood cell count nd leukocyte differentil count. Detils of the experimentl design re outlined in Figure 1. Sttisticl Anlysis A power nlysis performed using the SD from our preliminry studies confirmed tht 8 rts per group were sufficient for detecting differences etween the mens of the experiments, with power of Dt on CFU 308 Downloded from y guest on 01/15/2019 The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No. 5

4 Nsl infection, CFU Control, shm therpy, or LPT followed y 40 mg/kg PBS or levofloxcin Control, shm therpy, or LPT followed y 40 mg/kg PBS or levofloxcin; collect lood, lungs, nd spleens Control, shm therpy, or LPT followed y 40 mg/kg levofloxcin or PBS; collect lood, lungs, nd spleens Collect lood, lungs, nd spleens Time, h Figure 1. Experimentl protocol. On dy 0, rts were intrnslly infected with Streptococcus pneumonie colony-forming units (CFU) nd rndomly ssigned into control (n=8), shm therpy (n=8), or lymphtic pump tretment (LPT) (n=8) groups. At 24, 48, nd 72 hours fter infection, rts received control, shm therpy, or LPT followed y sucutneous injection of 40 mg/kg of levofloxcin or sterile phosphteuffered sline (PBS). S pneumonie CFU were enumerted t 48, 72, nd 96 hours fter infection. LPT or shm therpy ws dministered t 24 nd 48 hours fter infection, nd rts whose lungs nd spleens were collected t 48 hours did not receive intervention t 48 hours. LPT or shm therpy ws dministered t 24, 48, nd 72 hours fter infection, nd rts whose lungs nd spleens were collected t 72 hours did not receive intervention t 72 hours. were logrithmiclly trnsformed efore nlysis. Dt from control, shm therpy, or LPT were nlyzed y n nlysis of vrince followed y Tukey post hoc test using Grphpd Prism version 5.0 for Windows (Grph- Pd Softwre). Differences mong men vlues with P<.05 were considered sttisticlly significnt. Results Eight rts were in ech group, s follows: control, shm therpy, nd LPT, nd euthnized t 48 hours; control, shm therpy, nd LPT, nd euthnized t 72 hours; nd control, shm therpy, nd LPT, nd euthnized t 96 hours. In ddition, ech of the groups y euthniztion time received either PBS or levofloxcin efore euthniztion. Therefore, totl of 144 rts were used in the present study. Dt re presented s men (SD) unless otherwise indicted. LPT Protects Aginst Bcteril Pneumoni in Dose-Dependent Mnner Three pplictions of LPT significntly reduced the concentrtion of pulmonry cteri compred with control nd shm therpy (P<.05) (Figure 2). Additionlly, rts did not hve ny CFU in their spleens (dt not shown). LPT s n Adjunctive Therpy for Levofloxcin Levofloxcin is fluoroquinolone tht is commonly used s first choice for the mngement of CAP. 3,22 Levofloxcin reduced S pneumonie cteri in dose-dependent mnner over time (Figure 3). At 72 nd 96 hours fter infection, oth 40 mg/kg nd 50 mg/kg of levofloxcin significntly reduced CFU compred with PBS nd 25 mg/kg of levofloxcin (P<.05). At 96 hours fter infection, no CFU were detectle in the group receiving 50 mg/kg of levofloxcin. Considering tht no CFU were detectle in the 50-mg/kg levofloxcin group t 96 hours fter infection nd tht the protective effect of LPT ws not detectle until 96 hours fter infection (Figure 2), we chose 40 mg/kg of levofloxcin s the tretment dose for this study. At 48 hours fter infection, no significnt differences were noted in pulmonry CFU concentrtion etween the control plus PBS ( [ ]), shm therpy plus PBS ( [ ]), nd LPT plus PBS The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No Downloded from y guest on 01/15/2019

5 S pneumonie, Lung CFU S pneumonie, Lung CFU Infection Control Shm Therpy LPT Time, h Figure 2. Lymphtic pump tretment (LPT) protects ginst cteril pneumoni. Streptococcus pneumonie colony-forming units (CFU) were enumerted t 48, 72, nd 96 hours fter infection in rt model. P<.05 compred with control nd shm therpy. PBS mg/kg 40 mg/kg 50 mg/kg Time, h Figure 3. Levofloxcin dosing. On dy 0, rts were nslly infected with Streptococcus pneumonie colony-forming units (CFU) nd received phosphteuffered sline (PBS) nd 25 mg/kg (n=5), 40 mg/ kg (n=5), or 50 mg/kg (n=5) of levofloxcin t 24, 48, nd 72 hours fter infection. S pneumonie CFU were enumerted t 48, 72, nd 96 hours fter infection. *P<.05 compred with shm therpy nd control. ( [ ]) groups (P>.05). The ddition of levofloxcin significntly reduced S pneumonie CFU in the lungs compred with ll PBS groups (P<.05); however, no sttisticl differences were found etween the control plus levofloxcin ( [ ]), shm therpy plus levofloxcin ( [ ]), nd LPT plus levofloxcin ( [ ]) groups. Furthermore, CFU were not detected in spleens. These results re summrized in Figure 4A. Similrly, t 72 hours fter infection, no significnt differences were noted in pulmonry CFU etween the control plus PBS ( [ ]), shm therpy plus PBS ( [ ]), or LPT plus PBS ( [ ]) groups (P>.05). As expected, the ddition of levofloxcin significntly reduced S pneumonie CFU in the lungs compred with ll of the PBS groups (P<.05). There were significntly fewer cteri in shm therpy plus levofloxcin ( [ ]) nd LPT plus levofloxcin ( [ ]) groups compred with the control plus levofloxcin group ( [ ]) (P<.05). Furthermore, CFU were not detected in spleens. These results re summrized in Figure 4B. At 96 hours fter infection, the numer of CFU in the LPT plus PBS group ( [ ]) were significntly reduced compred with the control plus PBS group ( [ ]) (P<.05). Although the ddition of levofloxcin significntly reduced S pneumonie CFU in the lungs compred with ll PBS groups (P<.05), the dministrtion of LPT plus levofloxcin (531 [261]) significntly reduced cteril lod compred with ll intervention groups (P<.05) (Figure 4C). Furthermore, CFU were not detected in spleens (dt not shown), At 96 hours fter infection, ll of the rts in the control plus PBS nd shm therpy plus PBS groups hd disese (8 of 8 rts hd S pneumonie in their lungs). Rts in the control plus levofloxcin (2 of 8), shm therpy plus levofloxcin (3 of 8), nd LPT plus PBS (1 of 8) groups were modertely disese free; however, more thn hlf (5 of 8) of the rts in the LPT plus levofloxcin group were disese free. These results re summrized in Tle Downloded from y guest on 01/15/2019 The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No. 5

6 10 8 pneumonie, Lung CFU S pneumonie, Lung CFU AS B 10 8 Control Shm Therpy LPT Control Shm Therpy LPT PBS Levofloxcin Figure 4. Pulmonry Streptococcus pneumonie levels (A) 48, (B) 72, nd (C) 96 hours fter infection. At ech time point, rts were euthnized nd colony-forming units (CFU) were enumerted. During intervention sessions t 24 hours; 24 nd 48 hours; nd t 24, 48, nd 72 hours, respectively, rts received no therpy (control), shm therpy, or lymphtic pump tretment (LPT) followed y sucutneous injection of 40 mg/kg levofloxcin or sterile phosphteuffered sline (PBS). P<.05 compred with respective PBS injection groups. P<.05 compred with control + levofloxcin groups. c P<.05 compred with control + PBS injection groups d P<.05 compred with ll other intervention groups Control Shm Therpy LPT Control Shm Therpy LPT PBS Levofloxcin C 10 8 Tle 1. Disese Sttus 96 Hours After Infection With Streptococcus pneumonie in Rt Model S pneumonie, Lung CFU c d Intervention Disese Free, % LPT + PBS 13 Shm therpy + PBS 0 Control + PBS 0 LPT + levofloxcin 63 Shm therpy + levofloxcin 38 Control + levofloxcin Control Shm Therpy LPT Control Shm Therpy LPT PBS Levofloxcin Intervention Group The control group did not receive ny mnul therpy. Exceeded the upper limit y nlysis of mens for proportions. Arevitions: LPT, lymphtic pump tretment; PBS, phosphte-uffered sline. The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No Downloded from y guest on 01/15/2019

7 Effect of LPT on Hemtologic Fctors Hemtologic fctors were within the norml rnge for rts t ll time points mesured. The differences in levels etween seline nd 24 hours fter infection re summrized in Tle 2. At 48, 72, nd 96 hours fter infection, no sttisticlly significnt differences were found in the complete lood cell count or leukocyte differentil count etween ny of the intervention groups (Tle 3, Tle 4, nd Tle 5). Tle 2. Hemtologic Test Results t Bseline nd 24 h After Infection With Streptococcus pneumonie in Rt Model Test Result Bseline 24 h WBC, 10 6 cells/ml 5.3 (0.4) 4.6 (0.2) Neutrophil, 10 6 cells/ml 1.9 (0.2) 1.8 (0.1) Lymphocyte, 10 6 cells/ml 3.0 (0.2) 2.5 (0.1) Monocyte, 10 6 cells/ml 0.4 (0.04) 0.2 (0.01) Eosinophil, 10 6 cells/ml 0.01 (0.007) 0.02 (0.008) Bsophil, 10 6 cells/ml 0.01 (0.005) 0.01 (0.005) RBC, 10 6 cells/ml 8.4 (0.1) 9.0 (0.1) Hemogloin, g/dl 13.0 (0.3) 14.5 (0.2) Hemtocrit, % 48.7 (0.8) 52.2 (0.6) MCV, fl 58.4 (0.2) 58.1 (0.3) MCH, pg 15.5 (0.1) 16.2 (0.2) MCHC, g/dl 26.6 (0.2) 27.8 (0.3) RDW, % 15.8 (0.1) 14.1 (0.1) Pltelet, 10 8 cells/ml 7.5 (1.0) 4.9 (0.2) MPV, fl 5.8 (0.1) 6.3 (0.1) colony-forming units of S pneumonie. Dt were nlyzed y n nlysis of vrince, followed y Tukey post hoc test, nd re presented s men (SD). Arevitions: MCH, men corpusculr hemogloin; MCHC, men corpusculr hemogloin concentrtion; MCV, men corpusculr volume; MPV, men pltelet volume; RBC, red lood cell; RDW, red lood cell distriution width; WBC, white lood cell. Discussion The current study found tht when pplied once dily for 3 consecutive dys, the comintion of levofloxcin nd LPT significntly reduced the numer of S pneumonie cteri in the lungs compred with levofloxcin or LPT lone (P<.05). The multicenter osteopthic study in the elderly (MOPSE), doule-linded rndomized controlled tril, mesured the efficcy of osteopthic mnipultion s n djunctive tretment for hospitlized elderly ptients with pneumoni. 8,9 Within 24 hours of dmission, ptients were rndomly ssigned into conventionl cre (including ntiiotics), conventionl cre plus light touch, or conventionl cre plus OMT (including LPT). An intention-to-tret nlysis found no sttisticlly significnt difference etween the groups for ny outcome; however, perprotocol nlysis found tht OMT plus conventionl cre reduced the length of hospitl sty nd the durtion of intrvenous ntiiotic use nd decresed the incidence of respirtory filure or deth compred with the conventionl cre group lone. 9 Although the results from the MOPSE study support the use of OMT s n djunctive therpy for the tretment of ptients with pneumoni, the mechnism responsile for this protection is not cler. The results from the current study support the findings from the MOPSE study nd suggest tht LPT my protect ginst pneumoni y removing cteri from the lungs nd enhncing the efficcy of ntiiotics. In the current study, the comintion of shm therpy nd levofloxcin decresed cteril lod t 72 nd 96 hours fter infection. It is possile tht the propofol nesthesi dministered during shm therpy nd LPT enhnced pulmonry protection. Propofol hs een shown to protect ginst cute lung injury in rts y rogting the microvsculr lekge of wter nd protein in the lungs 23 nd suppressing inflmmtory-medited injuries. 24 Also, light touch my hve enhnced protection ginst infection, lthough this mechnism is less cler. Importntly, LPT plus levofloxcin clered more 312 Downloded from y guest on 01/15/2019 The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No. 5

8 Tle 3. Hemtologic Test Results 48 h After Infection With Streptococcus pneumonie in Rt Model PBS Levofloxcin Test Result Control Shm Therpy LPT Control Shm Therpy LPT WBC, 10 6 cells/ml 3.3 (0.4) 3.2 (0.6) 3.2 (0.5) 3.8 (0.4) 4.0 (0.4) 4.2 (0.2) Neutrophil, 10 6 cells/ml 1.2 (0.1) 1.3 (0.2) 1.3 (0.2) 1.3 (0.1) 1.5 (0.2) 1.5 (0.07) Lymphocyte, 10 6 cells/ml 2.0 (0.3) 1.8 (0.3) 1.7 (0.2) 2.3 (0.3) 2.3 (0.2) 2.5 (0.2) Monocyte, 10 6 cells/ml 0.1 (0.03) 0.1 (0.02) 0.1 (0.03) 0.1 (0.02) 0.2 (0.02) 0.2 (0.02) Eosinophil, 10 5 cells/ml 0.03 (0.02) 0.06 (0.03) 0.1 (0.05) 0.08 (0.04) 0.1 (0.06) 0.1 (0.04) Bsophil, 10 5 cells/ml 0.02 (0.02) 0.01 (0.01) 0.04 (0.02) 0.03 (0.02) 0.08 (0.04) 0.08 (0.04) RBC, 10 9 cells/ml 8.4 (0.3) 8.1 (0.2) 8.0 (0.5) 8.3 (0.4) 8.3 (0.3) 8.0 (0.3) Hemogloin, g/dl 13.5 (0.5) 13.2 (0.4) 12.7 (0.8) 12.8 (0.7) 12.8 (0.5) 13.5 (0.3) Hemtocrit, % 49.8 (1.9) 48.2 (1.5) 46.9 (2.9) 47.4 (2.6) 46.9 (1.9) 49.1 (1.0) MCV, fl 59.1 (0.5) 59.3 (0.6) 58.8 (0.4) 57.1 (0.6) 56.5 (0.4) 58.1 (1.1) MCH, pg 16.0 (0.1) 16.2 (0.2) 15.9 (0.1) 15.5 (0.3) 15.4 (0.1) 15.7 (0.3) MCHC, g/dl 27.1 (0.3) 27.1 (0.2) 27.1 (0.2) 27.1 (0.3) 31.0 (3.9) 27.6 (0.4) RDW, % 13.9 (0.1) 13.9 (0.1) 13.6 (0.2) 14.6 (0.2) 14.6 (0.2) 14.8 (0.3) Pltelet, 10 8 cells/ml 2.8 (0.9) 1.8 (0.7) 2.0 (0.9) 3.6 (0.7) 4.3 (0.6) 4.8 (0.6) MPV, fl 8.6 (1.1) 8.8 (0.9) 7.3 (0.5) 7.2 (0.5) 6.9 (0.6) 6.4 (0.1) colony-forming units of S pneumonie. Eight rts were in ech control, shm therpy, nd lymphtic pump tretment (LPT) group. Dt were nlyzed y n nlysis of vrince, followed y Tukey post hoc test, nd re presented s men (SD). Arevitions: MCH, men corpusculr hemogloin; MCHC, men corpusculr hemogloin concentrtion; MCV, men corpusculr volume; MPV, men pltelet volume; PBS, phosphte-uffered sline; RBC, red lood cell; RDW, red lood cell distriution width; WBC, white lood cell. cteri compred with shm therpy plus levofloxcin t oth time points, suggesting tht LPT plus levofloxcin induces n dditionl protective mechnism compred with shm therpy plus levofloxcin. We did not identify the mechnism y which LPT reduced cteri in the lungs, which is limittion. It is possile tht LPT my enhnce the delivery of levofloxcin to the lung. In support, the phrmcodynmic pttern of levofloxcin is linked to the clinicl outcome, 22 nd enhncing the delivery of levofloxcin to the lung vi LPT would support its use s n djunctive therpy. Furthermore, LPT hs een shown to enhnce the uptke of protein from the interstitil spce nd its trnsport to the lood 19 ; therefore, LPT my hve incresed protection ginst S pneumonie y enhncing the uptke of levofloxcin from the tissue nd its delivery to the lood. In the current study, levofloxcin ws dministered immeditely fter shm therpy or LPT; therefore, it is importnt to mention tht the protective effect of LPT my hve een gretly enhnced hd levofloxcin een dministered efore LPT. Nonetheless, further experimenttion is necessry to identify the mechnisms y which LPT cts s n djunctive therpy in this reserch model. The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No Downloded from y guest on 01/15/2019

9 Tle 4. Hemtologic Test Results 72 h After Infection With Streptococcus pneumonie in Rt Model PBS Levofloxcin Test Result Control Shm Therpy LPT Control Shm Therpy LPT WBC, 10 6 cells/ml 5.3 (0.4) 5.5 (0.4) 5.3 (0.3) 4.1 (0.5) 4.0 (0.4) 3.9 (.05) Neutrophil, 10 6 cells/ml 1.9 (0.2) 2.1 (0.2) 2.1 (0.2) 1.2 (0.09) 1.4 (0.1) 1.3 (0.2) Lymphocyte, 10 6 cells/ml 3.1 (0.2) 3.2 (0.2) 2.9 (0.2) 2.6 (0.3) 2.4 (0.3) 2.4 (0.3) Monocyte, 10 6 cells/ml 0.2 (0.03) 0.3 (0.02) 0.2 (0.03) 0.2 (0.03) 0.2 (0.03) 0.2 (0.03) Eosinophil, 10 5 cells/ml 0.2 (0.1) 0.08 (0.04) 0.2 (0.08) 0.1 (0.02) 0.06 (0.02) 0.1 (0.06) Bsophil, 10 5 cells/ml 0.1 (0.06) 0.05 (0.04) 0.1 (0.05) 0.04 (0.02) 0.03 (0.02) 0.05 (0.03) RBC, 10 9 cells/ml 9.0 (0.2) 8.7 (0.1) 8.7 (0.1) 8.5 (0.3) 8.3 (0.4) 7.8 (0.4) Hemogloin, g/dl 13.9 (0.3) 13.8 (0.2) 13.6 (0.3) 13.0 (0.3) 12.8 (0.6) 12.2 (0.6) Hemtocrit, % 51.9 (0.9) 51.1 (0.7) 50.4 (0.7) 48.7 (1.2) 47.2 (2.3) 44.4 (2.2) MCV, fl 57.9 (0.3) 58.4 (0.4) 58.1 (0.3) 57.5 (1.1) 56.5 (0.3) 57.3 (0.4) MCH, pg 15.4 (0.2) 15.8 (0.1) 15.7 (0.2) 15.4 (0.2) 15.0 (0.3) 15.8 (0.5) MCHC, g/dl 26.7 (0.2) 27.0 (0.2) 27.0 (0.4) 26.8 (0.2) 27.1 (0.3) 27.7 (1.0) RDW, % 14.2 (0.1) 14.1 (0.1) 14.1 (0.1) 16.1 (1.3) 14.9 (0.2) 14.7 (0.2) Pltelet, 10 8 cells/ml 4.6 (0.8) 6.4 (0.3) 5.9 (0.7) 4.8 (0.5) 4.4 (0.8) 5.1 (0.7) MPV, fl 6.6 (0.3) 6.2 (0.2) 6.5 (0.2) 6.5 (0.1) 7.0 (0.4) 6.9 (0.6) colony-forming units of S pneumonie. Eight rts were in ech control, shm therpy, nd lymphtic pump tretment (LPT) group. Dt were nlyzed y n nlysis of vrince, followed y Tukey post hoc test, nd re presented s men (SD). Arevitions: MCH, men corpusculr hemogloin; MCHC, men corpusculr hemogloin concentrtion; MCV, men corpusculr volume; MPV, men pltelet volume; PBS, phosphte-uffered sline; RBC, red lood cell; RDW, red lood cell distriution width; WBC, white lood cell. It is lso possile tht LPT enhnced pulmonry immunity. Studies in nd demonstrted tht LPT enhnced thorcic nd mesenteric lymph flow, moilized leukocytes from the gstrointestinl lymphoid tissues into lymph circultion, 12 nd incresed the lymphtic flux of leukocytes, cytokines, nd rective oxygen nd nitrogen species. 17,18 Collectively, these studies suggest tht LPT cn stimulte the lymphtic nd immune systems, which my ccelerte the removl of pneumoni y the immune system. In support, lymph cn ctivte leukocytes, 25 increse endothelil cell permeility, 26,27 nd redistriute leukocytes, cyto- kines, nd chemokines to the lung. 28 If the lung is infected, mesures tht enhnce lymph output, such s LPT, my ccelerte the immune-medited clernce of the microes; however, whether LPT enhnces this process is unknown. Conclusion The comintion of levofloxcin nd LPT significntly reduced the concentrtion of pulmonry cteri compred with LPT or levofloxcin lone (P<.05). To our knowledge, this study is the first to demonstrte tht 314 Downloded from y guest on 01/15/2019 The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No. 5

10 Tle 5. Hemtologic Test Results t Bseline nd 96 h After Infection With Streptococcus pneumonie in Rt Model PBS Levofloxcin Test Result Control Shm Therpy LPT Control Shm Therpy LPT WBC, 10 6 cells/ml 5.8 (0.4) 5.1 (0.4) 5.2 (0.4) 4.5 (0.4) 4.5 (0.3) 4.7 (0.4) Neutrophil, 10 6 cells/ml 2.5 (0.2) 2.0 (0.2) 1.9 (0.2) 1.7 (0.2) 1.8 (0.2) 1.9 (0.2) Lymphocyte, 10 6 cells/ml 3.0 (0.2) 2.8 (0.2) 3.0 (0.3) 2.5 (0.3) 2.4 (0.2) 2.6 (0.2) Monocyte, 10 6 cells/ml 0.2 (0.03) 0.3 (0.02) 0.3 (0.04) 0.3 (0.03) 0.3 (0.03) 0.3 (0.03) Eosinophil, 10 5 cells/ml 0.01 (0.003) 0.02 (0.005) (0.003) 0.01 (0.01) (0.002) (0.003) Bsophil, 10 5 cells/ml (0.002) 0.01 (0.003) (0.002) (0.006) (0.002) (0.002) RBC, 10 9 cells/ml 7.8 (1.1) 7.9 (0.4) 7.9 (1.1) 8.4 (0.4) 7.8 (0.3) 8.0 (0.4) Hemogloin, g/dl 14.1 (0.1) 12.5 (0.7) 13.7 (0.3) 13.3 (0.7) 12.3 (0.4) 12.5 (0.7) Hemtocrit, % 51.0 (0.6) 45.7 (2.4) 51.5 (1.0) 47.9 (2.2) 44.5 (1.4) 45.1 (2.1) MCV, fl 57.4 (0.4) 57.9 (0.4) 59.0 (0.9) 56.7 (0.2) 56.9 (0.2) 56.9 (0.3) MCH, pg 15.9 (0.1) 15.8 (0.2) 15.8 (0.1) 15.7 (0.2) 15.6 (0.2) 15.7 (0.2) MCHC, g/dl 27.6 (0.3) 27.3 (0.3) 27.3 (0.2) 27.6 (0.3) 27.5 (0.4) 27.5 (0.4) RDW, % 14.7 (0.3) 14.6 (0.2) 14.9 (0.3) 15.7 (0.2) 15.4 (0.2) 15.2 (0.2) Pltelet, 10 8 cells/ml 7.3 (0.8) 6.2 (0.7) 9.0 (1.9) 7.0 (0.8) 6.6 (0.7) 7.6 (0.5) MPV, fl 6.1 (0.1) 6.3 (0.1) 6.0 (0.1) 6.1 (0.1) 6.2 (0.1) 5.8 (0.1) colony-forming units of S pneumonie. Eight rts were in ech control, shm therpy, nd lymphtic pump tretment (LPT) group. Dt were nlyzed y n nlysis of vrince, followed y Tukey post hoc test, nd re presented s men (SD). Arevitions: MCH, men corpusculr hemogloin; MCHC, men corpusculr hemogloin concentrtion; MCV, men corpusculr volume; MPV, men pltelet volume; PBS, phosphte-uffered sline; RBC, red lood cell; RDW, red lood cell distriution width; WBC, white lood cell. LPT cts synergisticlly with ntiiotics for the tretment of ptients with pneumoni. Trnsltionl studies such s the current study re crucil to identify the mechnisms y which LPT protects ginst infectious disese nd to support its clinicl use in ptients with pneumoni. Once these mechnisms re understood, LPT cn e optimlly pplied to ptients with pneumoni, which my sustntilly reduce moridity, mortlity, nd the cost of hospitliztion. Author Contriutions All uthors provided sustntil contriutions to conception nd design, cquisition of dt, or nlysis nd interprettion of dt; ll uthors drfted the rticle or revised it criticlly for importnt intellectul content; Dr Hodge gve finl pprovl of the version of the rticle to e pulished; nd ll uthors gree to e ccountle for ll spects of the work in ensuring tht questions relted to the ccurcy or integrity of ny prt of the work re ppropritely investigted nd resolved. (continued) The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No Downloded from y guest on 01/15/2019

11 References 1. Brr NK, Niedermn MS. Mngement of community cquired pneumoni: review nd updte. Ther Adv Respir Dis. 2011;5(1): doi: / Hll MJ, DeFrnces CJ, Willims SN, Golosinskiy A, Schwrtzmn A. Ntionl Hospitl Dischrge Survey: 2007 summry. Ntl Helth Stt Rep. 2010;26(29):1-20, Restrepo MI, Frei CR. Helth economics of use fluoroquinolones to tret ptients with community-cquired pneumoni. Am J Med. 2010;123(4 suppl):s39-s46. doi: /j.mjmed Kucher ML. Lymphtics. In: Chil AG, executive ed. Foundtions of Osteopthic Medicine. 3rd ed. Phildelphi, PA: Lippincott Willims nd Wilkins; 2011: Degenhrdt BF, Kucher ML. Updte on osteopthic medicl concepts nd the lymphtic system. J Am Osteopth Assoc. 1996:96(2): Jckson KM, Steele TF, Dugn EP, Kukulk G, Blue W, Roerts A. Effect of lymphtic nd splenic pump techniques on the ntiody response to heptitis B vccine: pilot study. J Am Osteopth Assoc. 1998:98(3): Mesel JW Jr. The effect of lymphtic pump on the immune response, I: preliminry studies on the ntiody response to pneumococcl polyscchride ssyed y cteril gglutintion nd pssive hemgglutintion. J Am Osteopth Assoc. 1982:82(1): Noll DR, Shores JH, Gmer RG, Herron KM, Swift J Jr. Benefits of osteopthic mnipultive tretment for hospitlized elderly ptients with pneumoni. J Am Osteopth Assoc. 2000;100(12): Noll DR, Degenhrdt BF, Morely TF, et l. Efficcy of osteopthic mnipultion s n djunctive tretment for hospitlized ptients with pneumoni: rndomized controlled tril. Osteopth Med Prim Cre. 2010;4:2. doi: / Allen TW, Pence TK. The use of the thorcic pump in tretment of lower respirtory trct disese. J Am Osteopth Assoc. 1967;67(4): Hodge LM, King HH, Willims AG, et l. Adominl lymphtic pump tretment increses leukocyte count nd flux in thorcic duct lymph. Lympht Res Biol. 2007;5(2): Hodge LM, Berden MK, Schnder A, et l. Adominl lymphtic pump tretment moilizes leukocytes from the gstrointestinl ssocited lymphoid tissue into lymph. Lympht Res Biol. 2010;8(2): doi: /lr Hodge LM, Downey HF. Lymphtic pump tretment enhnces the lymphtic nd immune systems. Exp Biol Med. 2011;236(10): doi: /em Hodge LM. Osteopthic lymphtic pump techniques to enhnce immunity nd tret pneumoni. Int J Osteopth Med. 2012;15(1): Huff JB, Schnder A, Downey HF, Hodge LM. Lymphtic pump tretment ugments lymphtic flux of lymphocytes in rts. Lympht Res Biol. 2010;8(4): doi: /lr Knott EM, Tune JD, Stoll ST, Downey HF. Incresed lymphtic flow in the thorcic duct during mnipultive intervention. J Am Osteopth Assoc. 2005;105(10): Schnder A, Downey HF, Hodge LM. Lymphtic pump mnipultion moilizes inflmmtory meditors into lymphtic circultion. J Experimentl Biol Med. 2012;237(1): doi: /em Schnder A, Pdro D, King HH, Downey HF, Hodge LM. Lymphtic pump tretment repetedly enhnces the lymphtic nd immune systems. Lympht Res Biol. 2013;11(4): doi: /lr Dery MA, Yonuschot G, Winterson BJ. The effects of mnully pplied intermittent pulstion pressure to rt ventrl thorx on lymph trnsport. Lymphology. 2000;33(2): Cresy C, Schnder A, Orlowski A, Hodge LM. Thorcic nd dominl lymphtic pump techniques inhiit the growth of S. pneumonie cteri in the lungs of rts. Lympht Res Biol. 2013;11(3): doi: /lr Institutionl Animl Cre nd Use Committee. Guide for the Cre nd Use of Lortory Animls. 8th ed. Wshington, DC: The Ntionl Acdemy of Sciences; Alertson TE, Den NC, El Solh AA, et l. Fluoroquinolones in the mngement of community cquired pneumoni [review]. Int J Clin Prct. 2010;64(3): doi: /j x. 23. Chu CH, Dvid Liu D, Hsu YH, Lee KC, Chen HI. Propofol exerts protective effects on the cute lung injury induced y endotoxin in rts. Pulm Phrmcol Ther. 2007;20(5): Chn KC, Lin CJ, Lee PH, et l. Propofol ttenutes the decrese of dynmic complince nd wter content in the lung y decresing oxidtive rdicls relesed from the reperfused liver. Anesth Anlg. 2008;107(4): doi: /ne.0013e318181f4e Cruso JM, Feketeov E, Dyl SD, Huser CJ, Ditch EA. Fctors in intestinl lymph fter shock increse neutrophil dhesion molecule expression nd pulmonry leukosequestrtion. J Trum. 2003;55(4): Deitch EA, Adms CA, Lu Q, nd Xu DZ. Mesenteric lymph from rts sujected to trum-hemorrhgic shock re injurious to rt pulmonry microvsculr endothelil cells s well s humn umilicl vein endothelil cells. Shock. 2001;16(4): Breithupt-Flopp AC, Vitoretti LB, Cvrini G, et l. Intestinl lymph-orne fctors induce lung relese of inflmmtory meditors nd expression of dhesion molecules fter n intestinl ischemic insult. J Surg Res. 2012;176(1): doi: /j.jss Dvidson MT, Deitch EA, Lu Q, et l. A study of the iologic ctivity of trum-hemorrhgic shock mesenteric lymph over time nd the reltive role of cytokines. Surgery. 2004;136(1): Americn Osteopthic Assocition 316 Downloded from y guest on 01/15/2019 The Journl of the Americn Osteopthic Assocition My 2015 Vol 115 No. 5