Subiliac Lymph Nodes Research Articles

Preliminary Study on the Effective Microbial Supplementation of Feed on the Infection of <i>Salmonella</i> in Two Lymph Nodes of Beef Cattle in Eastern Ethiopia

A double-blinded randomized controlled field trial based on parallel group design was conducted from January, 2018 to July, 2018 in Chercher Oda-Bultum Farmers Union beef Farm. The present study was conducted to evaluate the roll of effective microbial supplementation to feed on the infection of Salmonella in the mesenteric and sub-iliac lymph nodes of beef cattle. In order to undertake the study, 130 beef cattle kept by the farm were used to establish a cohort. The study animals were randomly assigned to the treatment group (n = 100) and control group (n = 30). The feed of treatment group was mixed with EM at dose of 5× 1010 cfu/day/head for 90, 100 and 115 days while that of the control group was mixed with molasses, which acts as placebo. Both the treatment and control were slaughtered and two lymph nodes were collected from each animal under strict sterile condition and processed for the isolation and identification of Salmonella using standard procedure. The occurrence of Salmonella was 70% (CI = 51% - 85%) in control group while it was 33% (CI = 24% - 43%) in treatment group. The difference in the proportion of Salmonella infection in the two group was significant (x2 = 13.01; p = 0.000). The relative risk of Salmonella isolation in the control was 2.12 (1.41 - 3.20) compared to treatment group. The absolute and relative risk reduction in the treatment were 37% (CI = 17% - 57%) and 53% (CI = 29% - 69%), respectively. This preliminary study indicated that effective microbial supplementation of beef cattle feed reduced the occurrences of Salmonella in the lymph node of beef cattle, thereby potentially minimizing the economic and public health impacts of Salmonella infection. Then, it was recommended to use EM as prevention and control option in Salmonella carriage in cattle.

Evaluating the Effective Microbial Supplementation of Feed on the Load of <i>Salmonella</i> in Two Lymph Nodes of Beef Cattle in Eastern Ethiopia

The present study was conducted to evaluate the role of effective microbial supplementation to feed on the load of Salmonella in the mesenteric and sub-iliac lymph nodes of beef cattle. Bulls of Harer cattle breed managed at Chercher Oda-Bultum Farmers Union beef Farm were used as study subject. A total of 130 bulls were used using double blinded randomized controlled field trial based on parallel group design from January 2018 to July 2018. The study animals were randomly assigned to the treatment group (n = 100) and control group (n = 30). The feed of treatment group was mixed with EM at dose of 5 × 1010 cfu/day/head and supplemented for 90, 100 and 115 days while that of the control group was mixed with molasses, which acts as placebo. Both the treatment and control were slaughtered and two lymph nodes were collected from each animal under strict sterile condition and processed for the isolation and identification of Salmonella using standard procedure. A significant (p = 0.001) reduction in the load of Salmonella was observed in the lymph node of treatment group as compared to the control group. The load of Salmonella was significantly affected by length of feeding period and age of bulls. This study indicated that effective microbial supplementation to bulls from Harar cattle reduces the load of Salmonella in the lymph node of beef cattle thereby potentially minimizing the economic and public health impacts of Salmonella infection.

Establishment of anti-HBcAg Monoclonal Antibodies for Sandwich ELISA Application by Iliac Method Utilizing Incomplete Adjuvant

The prevalence of hepatitis B virus (HBV) infection in Indonesia was moderate in 2013. This makes an appropriate action has to be taken immediately. In an attempt to evaluate our candidate HBV vaccine efficacy, we generated monoclonal antibodies specific to HBcAg for sandwich ELISA application for research purposes. Monoclonal antibodies are generally developed using hybridoma technology by isolating B cells from spleen or lymph nodes followed by fusion with myeloma cells. This study generated hybridoma by modifying the iliac lymph node method and used incomplete Freund’s adjuvant to emulsify the antigen. For this purpose, an eleven-week-old BALB/c mouse was immunized with a single shot recombinant HBcAg water in oil emulsion intramuscularly at the mouse tail base. After one month, besides enlargement of the medial iliac lymph nodes, we also found that the sub-iliac lymph nodes were enlarged. In two different fusion attempts, the single B cells were then isolated from each lymph node and fused with SP2/0—Ag14 mouse myeloma cells. Hybridoma cells derived from both lymph nodes were screened by ELISA coated with HBcAg peptide. As a result, we obtained two positive wells of hybridoma polyclones from each medial iliac and sub-iliac-derived B lymphocyte. After monoclonalization, we obtained candidate anti-HBcAg monoclonal antibodies as either capture or detection antibodies. Furthermore, we successfully retrieved hybridoma clones 9.3, 9.4, and 9.5, which could produce monoclonal antibodies for sandwich ELISA application.

Abstract LBA048: Lymph node metastasis targeted intranodal delivery of docetaxel improves treatment outcome

Abstract Despite recent advances in diagnostic modalities and cancer treatment guidelines, systemic chemotherapy for the lymph node (LN) metastasis has poor treatment efficacy using conventional drugs. Therefore, a novel method, lymphatic drug delivery system (LDDS), has been proposed in a pre-clinical experiment that directly injects the anticancer drug into sentinel LNs to deliver into their downstream LNs, aiming to inhibit tumor cell growth in those LNs. The injection duration of chemotherapy drugs using the LDDS will be less than the intratumoral or intralesion methods used in the clinic, and systemic toxicity will be less than tumor-targeted therapy or systemic chemotherapy. Our previous results indicated that the treatment efficacy increased in LDDS treated groups than systemic delivery. Here, we show the improvement of treatment efficacy by increasing osmotic pressure and viscosity of the drug solvent delivered by LDDS using the metastatic LN mouse model. First, a metastatic LN mouse model was created by inoculating luciferase-expressing tumor cells into subiliac LN (SiLN) of MXH10/Mo/lpr mouse to induce metastasis into the proper axillary LN (PALN). Next, 10 mg/kg docetaxel (DTX) with increased osmotic pressure and viscosity were injected into tumor-inoculated SiLN with an injection rate of 2400 μL/min on day 7 post-inoculation to inhibit tumor cell growth in the inoculation and metastatic site. Luciferase activity decreases in PALNs and SiLNs were observed in groups between 1140 kPa, 1960 kPa, and 2780 kPa (4, 11, and 38 mPa·s) groups without inducing any side effects. By histology, no tumor cells were confirmed in those LNs of the 1960 kPa group. Then, DTX at 1960 kPa, 11 mPa·s was injected into tumor-inoculated SiLN on day 21 post-inoculation with different injection speeds and volumes. We found that DTX at 1960 kPa, 11 mPa·s injected at the injection rate of 200 μL/min with 400 μL volume, can inhibit tumor cell growth in the PALN and delay tumor growth SiLN. Furthermore, the antitumor effects depended on the tumor progression stage in the LNs, and the injection rates and volumes of administered drugs. We anticipate these optimal ranges as a starting point for developing more effective drug regimens to treat metastatic LNs with the LDDS. Moreover, administrable drugs in this novel method of ultrasound-guided LDDS or LDDS at intra-operative or image-guided surgery are not limited only to anticancer agents. Citation Format: Ariunbuyan Sukhbaatar, Shouta Sora, Mori Shiro, Tetsuya Kodama. Lymph node metastasis targeted intranodal delivery of docetaxel improves treatment outcome [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA048.

Abstract LBA049: Possibility of total body irradiations for lymph node metastasis and lung metastasis

Abstract Metastatic lymph nodes (MLNs) are one of the most common causes of distant metastasis, and their presence signals a serious deterioration in the life prognosis of an affected patient. The treatment of MLNs at an early stage is necessary to inhibit metastases to distant LNs and organs. Radiation therapy is an effective therapeutic modality for local tumor control in many cancers, and its effect is enhanced by fractionated radiation therapy or in combination with other therapeutic regimens. In addition, recent studies have reported that total body irradiation (TBI) increases the activation of immune cells such as NK cells and infiltration of CD4+ T cells to tumor tissue. However, the effects of TBI on LN metastasis remains unclear. This study assessed the effect of TBI by changing the radiation dose and number of irradiations in an MLN mouse model. To induce metastasis into the proper axillary lymph node (PALN) and lung, mouse breast cancer FM3A-Luc cells were inoculated into the subiliac lymph node (SiLN) of MXH10/Mo/lpr mice. Mice were assigned to 4 groups thus: received no TBI; received a single dose of 0.2 Gy as low-dose TBI (single L-TBI); received a single dose of 1.0 Gy as middle-dose TBI (single M-TBI); or received two fractions of 2.0 Gy (fractionated M-TBI). The irradiation was carried out on day 4 post-inoculation in the single L-TBI and M-TBI groups. In the fractionated M-TBI group the irradiation was carried out on days 4 and 7 post-inoculation. Tumor growth was evaluated using an in vivo bioluminescence imaging system. In the non-irradiated group, tumor activity in the SiLN and PALN increased over time and lung metastasis was confirmed on day 28 post-inoculation. In addition, the single L-TBI dose did not delay tumor growth in the SiLN and PALN or inhibit lung metastasis. In other words, the tumor activity was not significantly changed compared to the non-irradiated group. Single and fractionated M-TBI doses significantly delayed tumor growth in the SiLN and PALN and inhibited lung metastasis. Furthermore, the fractionated M-TBI group exhibited the greatest delay in tumor growth in the PALN compared to the other three groups. These results suggest that the TBI at a dose > 1.0 Gy can delay tumor growth of MLNs in the early stage and distant metastasis. Moreover, the radiation dose and number of irradiations may be essential factors for enhancing the anti-tumor effects of TBI. We anticipate that TBI will contribute to the prevention and treatment of cancer metastasis via LNs. Citation Format: Shouta Sora, Radhika Mishra, Ariunbuyan Sukhbaatar, Maya Sakamoto, Shiro Mori, Tetsuya Kodama. Possibility of total body irradiations for lymph node metastasis and lung metastasis [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA049.

Abstract LBA051: Importance of drug osmotic pressure and viscosity for efficient drug delivery using lymphatic drug delivery system

Abstract Metastasis is known to be the prime cause of cancer associated fatality. It is therefore imperative to treat and prevent metastasis in order to curb cancer-associated morbidity and mortality. Metastatic lymph nodes (MLNs) are known to be crucial diagnostic and prognostic indicators. Furthermore, as per the lymph node mediated hematogenous theory, due to extant communication between the lymphatic network and the venous system, the lymphatic network facilitates the passage and subsequent colonization of tumor cells to distant sites, thus, serving as a starting point for distant metastasis even during the initial stages. Treatment of MLNs, therefore, is imperative. Proposed in 2015, the Lymphatic Drug Delivery System (LDDS) is a novel and promising drug delivery system for the treatment of MLNs that overcomes the limitations of conventional chemotherapy. Unlike systemic chemotherapy, LDDS entails a direct intranodal drug administration to sentinel lymph node, post its identification using radioisotopes and fluorescent molecules, in order to treat it and its downstream lymph nodes. Previous studies have documented the superior efficacy of LDDS as a MLN management strategy as compared to conventional therapy. Higher drug delivery to target sites and prolonged retention with lower systemic toxicity, and thereby stronger anti-tumor effect have been reported to have been achieved upon drug administration via LDDS. In the present study, MXH10/Mo/lpr mice and FM3A-Luc, mouse mammary carcinoma cells expressing the firefly luciferase gene, have been utilized for the establishment of a mouse model of Lymph node metastasis (LNM) to investigate the effect of drug osmotic pressure and viscosity on therapeutic efficacy of the LDDS. Two lymph nodes, exhibiting systemic lymphadenopathy, in the subiliac and axillary region, the subiliac lymph node (SiLN) and proper axillary lymph node (PALN) have been used to model a network of interconnected human lymph nodes. Drug solution of varying osmotic pressure and viscosity were administered via LDDS to the murine model of LNM and subsequently therapeutic efficacy was monitored using the high frequency ultrasound system, VEVO and bioluminescent imaging system, IVIS, and confirmed through histology. The study revealed an optimized window of drug osmotic pressure and viscosity (π = 1000 - 2000 kPa; µ = 0.9 - 11.5 mPa·s.) where a significantly stronger anti-tumor response was observed. Venturing below or beyond the optimized range results in decreased therapeutic efficacy. The study therefore concluded that drug osmotic pressure and viscosity are important fluid parameters that require careful consideration prior to preparation of drug cocktails as they can severely impact the prognosis. The findings of this study have the potential for translation to clinics and we anticipate that these results can be replicated in clinical trials, positively altering the fate of many cancer patients. Citation Format: Radhika Mishra, Ariunbuyan Sukhbaatar, Sora Shouta, Maya Sakamoto, Shiro Mori, Tetsuya Kodama. Importance of drug osmotic pressure and viscosity for efficient drug delivery using lymphatic drug delivery system [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA051.

The Therapeutic Effect of Second Near-Infrared Absorbing Gold Nanorods on Metastatic Lymph Nodes via Lymphatic Delivery System.

Photothermal therapy has been established recently as a non-invasive treatment protocol for cancer metastatic lymph nodes. Although this treatment approach shows efficient tumour ablation towards lymph node metastasis, the monitoring and reporting of treatment progress using the lymphatic delivery channel still need to be explored. Herein, we investigated the anti-tumour effect of pegylated gold nanorods with a high aspect ratio (PAuNRs) delivered via the lymphatic route in a mouse model. In this study, breast carcinoma (FM3A-Luc) cells were inoculated in the subiliac lymph node (SiLN) to induce metastasis in the proper axillary lymph node (PALN). The treatment was initiated by injecting the PAuNRs into the accessory axillary lymph node (AALN) after tumour metastasis was confirmed in the PALN followed by external NIR laser irradiation under a temperature-controlled cooling system. The anti-tumour impact of the treatment was evaluated using an in vivo bioluminescence imaging system (IVIS). The results showed a time-dependent reduction in tumour activity with significant treatment response. Tumour growth was inhibited in all mice treated with PAuNRs under laser irradiation; results were statistically significant (** p < 0.01) even after treatment was concluded on day 3. We believe that this non-invasive technique would provide more information on the dynamics of tumour therapy using the lymphatically administered route in preclinical studies.

159 Evaluation of Bacillus Subtilis PB6 Probiotic (CLOSTAT® 500) on Feedlot Phase Growth Performance, Efficiency of Dietary Net Energy Utilization, and Fecal and Subiliac Lymph Node Salmonella Prevalence

Abstract Yearling beef steers (n = 238; initial BW=402 ± 31.2 kg) were used to evaluate a Bacillus subtilis probiotic on growth performance, dietary net energy (NE) utilization, carcass characteristics, and fecal and subiliac lymph node Salmonella prevalence during a 140-d finishing period. Steers were allotted to 24 pens (n = 9 to 10 steers/pen) and assigned to one of two treatments (12 pens/treatment): no probiotic (CON) or 0.50 g·steer-1·d-1 of a Bacillus subtilis PB6 probiotic (CLOSTAT® 500, Kemin Industries, Des Moines, IA; CLO). Steers were transitioned to a 90% concentrate diet (DM basis) over 14-d. Steers were fed once daily at 0700 h; bunks were managed according to a slick bunk management. Fecal samples were collected on d 1, 28, 56, 112, and 140 from each pen (n = 5 steers/pen) via rectal palpation and composited by pen for determination of Salmonella prevalence. Upon harvest, subiliac lymph nodes were obtained from 60 steers in CON and 57 steers in CLO. Data were analyzed as a randomized complete block design; pen was the experimental unit; α of 0.05 determined significance. No differences were detected (P ≥ 0.25) between treatments for live or carcass-adjusted average daily gain, dry matter intake, gain efficiency, dietary NE utilization, nor calculated dietary NE content based upon performance. No differences were detected between treatments for any carcass traits (P ≥ 0.15). Salmonella was not recovered in any fecal samples except on d 112, where steers from CLO had a numerically lower (P = 0.17; 8.3 vs. 25.0%) incidence of fecal Salmonella compared to CON and on d 140 fecal, where Salmonella incidence did not differ (P = 0.34; 0.0 vs. 8.3%) for CON and CLO, respectively. Salmonella was not recovered in any subiliac lymph nodes. These data indicate that CLO did not influence growth performance or Salmonella prevalence.

Study of the physicochemical properties of drugs suitable for administration using a lymphatic drug delivery system.

Lymph node (LN) metastasis is thought to account for 20‐30% of deaths from head and neck cancer. The lymphatic drug delivery system (LDDS) is a new technology that enables the injection of drugs into a sentinel LN (SLN) during the early stage of tumor metastasis to treat the SLN and secondary metastatic LNs. However, the optimal physicochemical properties of the solvent used to carry the drug have not been determined. Here, we show that the osmotic pressure and viscosity of the solvent influenced the antitumor effect of cisplatin (CDDP) in a mouse model of LN metastasis. Tumor cells were inoculated into the proper axillary LN (PALN), and the LDDS was used to inject CDDP solution into the subiliac LN (SiLN) to treat the tumor cells in the downstream PALN. CDDP dissolved in saline had no therapeutic effects in the PALN after it was injected into the SiLN using the LDDS or into the tail vein (as a control). However, CDDP solution with an osmotic pressure of ~ 1,900 kPa and a viscosity of ~ 12 mPa⋅s suppressed tumor growth in the PALN after it was injected into the SiLN using the LDDS. The high osmotic pressure dilated the lymphatic vessels and sinuses to enhance drug flow in the PALN, and the high viscosity increased the retention of CDDP in the PALN. Our results demonstrate that optimizing the osmotic pressure and viscosity of the solvent can enhance the effects of CDDP, and possibly other anticancer drugs, after administration using the LDDS.

Evaluation of Bacillus subtilis PB6 on feedlot phase growth performance, efficiency of dietary net energy utilization, and fecal and subiliac lymph node Salmonella prevalence in spring placement yearling beef steers fed in southeastern South Dakota,.

Yearling crossbred beef steers [N = 238; initial shrunk body weight (BW) = 402 ± 31.2 kg] were used to investigate the influence of a Bacillus subtilis probiotic on animal growth performance, efficiency of dietary net energy (NE) utilization, carcass characteristics, and fecal and subiliac lymph node Salmonella prevalence during a 140-d finishing period at the Southeast Research Farm in Beresford, SD. Steers were allotted to 1 of 24 pens (N = 9–10 steers/pen) and assigned to 1 of 2 dietary treatments (12 pens/treatment): no probiotic (CON) or 0.5 g/steer/d of a B. subtilis PB6 probiotic (CLOSTAT500, Kemin Industries, Des Moines, IA; CLO). Bunks were managed according to a slick bunk management approach. Fecal samples were collected on study days 1, 28, 56, 112, and 140 from a subsample of steers from each pen (N = 5 steers/pen) via rectal palpation and composited by pen for the determination of Salmonella prevalence using selective enrichment and culture media. Upon harvest, subiliac lymph nodes were obtained from an equal number of steers from each treatment (collected from every other steer) following evisceration and hide removal. Data were analyzed as a randomized complete block design and pen served as the experimental unit; an α of 0.05 determined significance. Live-basis final BW and average daily gain tended (P ≤ 0.06) to be reduced for CLO. No differences were detected (P ≥ 0.11) between treatments for dry matter intake or gain efficiency. Treatment neither altered the efficiency of dietary NE utilization nor calculated dietary NE content based upon observed performance (P ≥ 0.46). No differences were detected between treatments for any carcass traits (P ≥ 0.15). No Salmonella was recovered in any fecal samples collected on study days 1, 28, or 56. On day 112, steers from CLO had a numerically lower (P = 0.17; 25.0 vs. 8.3%) incidence of fecal Salmonella compared to CON. On study day 140, fecal Salmonella incidence did not differ between treatments (P = 0.34; 0.0 vs. 8.3%) for CON and CLO, respectively. Upon harvest, no Salmonella was recovered in any subiliac lymph nodes. These data indicate that B. subtilis PB6 did not influence feedlot phase growth performance or fecal Salmonella prevalence. Additionally, Salmonella was not observed in the subiliac lymph nodes of any steers upon harvest.

Evaluation of Autogenous Vaccine Use in Mitigating Salmonella in Lymph Nodes from Feedlot Cattle in Texas

Managing the presence of Salmonella in ground beef has been an ongoing challenge for the beef industry. Salmonella prevalence can vary regionally, seasonally, and within the animal, making the development of interventions difficult. The objective of this study was to assess the efficacy of an autogenous Salmonella vaccine in mitigating Salmonella in lymph nodes (LNs) of feedlot cattle. An autogenous vaccine was developed using the most common Salmonella enterica serovars (Salmonella Kentucky, Salmonella Anatum, Salmonella Muenchen, Salmonella Montevideo, and Salmonella Mbandaka) identified from cattle managed at a South Texas feedlot with historically high Salmonella prevalence. Fifty-five heifers were selected for even distribution across five groups: (i) BASE, which received no autogenous vaccinations and were harvested after the stocker stage, (ii) CNTRL, which received no autogenous vaccinations, (iii) FARM, which received autogenous vaccinations at the ranch only, (iv) SPLIT, which received autogenous vaccinations at both the ranch and feedlot, and (v) YARD, which received vaccinations at the feedlot only. One heifer each from the BASE and CNTRL groups did not complete the study. All treatment groups except BASE were harvested after reaching market weight. Left and right superficial cervical and subiliac LNs from each carcass were collected and analyzed for Salmonella presence, and positive samples were serotyped. No salmonellae were recovered from LNs derived from BASE, FARM, SPLIT, or YARD groups. Cattle in the BASE group were expected to have a low occurrence of Salmonella based on previous research. However, the percentage of Salmonella-positive animals in the CNTRL group was 20.0% (2 of 10), which is lower than expected based on historical data from the same feeding location. There could be several causes of decreased Salmonella presence in the LNs of control cattle, creating an opportunity for future investigation into the development of preharvest interventions to combat Salmonella in feedlots.

Presence of Presumptive Shiga Toxin–Producing Escherichia coli and Salmonella on Sheep during Harvest in Honduras

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) and Salmonella are foodborne pathogens commonly harbored in the gastrointestinal tract of sheep. These pathogens can be on the hide of sheep and transferred to the carcass, causing a foodborne hazard. Salmonella can also be found in the lymph nodes of sheep, creating a biological hazard during harvest and processing. Developing countries lack baseline data on the presence of these foodborne pathogens on meat products, specifically sheep meat. Therefore, the objective of this study was to evaluate the presence of STEC and Salmonella on sheep hides, preevisceration carcasses, and final carcasses and Salmonella in subiliac lymph nodes from two small Honduran harvest facilities, plants A and B. Sponge swabs from the foreshank region of hides and carcasses and subiliac lymph node samples were collected from 96 sheep (86 at plant A; 10 at plant B). Microbial detection of STEC and Salmonella was performed by using the BAX System to screen for prevalence, and suspect samples were subjected to conventional culture isolation for confirmation. Overall baseline contamination on hides were 34.4 and 10.4% present for STEC and Salmonella; however, through implementation of sanitary procedures, preevisceration and final carcass samples were significantly decreased from the hide for both pathogens (P = 0.05). Moreover, overall plant A had significantly higher rates (P = 0.05) of STEC and Salmonella at each carcass sampling site compared with plant B. After each sampling was performed, recommendations were provided to each facility on the basis of pathogen presence, performance techniques, and contamination risks. Through recommendations and implementation of hazard analysis and critical control points, good manufacturing practices, and sanitation standard operating procedures in each facility, the meat supply in Honduras will become safer and more wholesome. Therefore, the development of a baseline with continued sampling is crucial to understand the risk of foodborne pathogens to consumers in the Honduran sheep meat supply. HIGHLIGHTS

Intranodal pressure of a metastatic lymph node reflects the response to lymphatic drug delivery system

Cancer metastasis to lymph nodes (LNs) almost certainly contributes to distant metastasis. Elevation of LN internal pressure (intranodal pressure, INP) during tumor proliferation is associated with a poor prognosis for patients. We have previously reported that a lymphatic drug delivery system (LDDS) allows the direct delivery of anticancer drugs into the lymphatic system and is a promising treatment strategy for early‐stage LN metastasis. However, methods for evaluating the treatment effects have not been established. Here, we used a mouse model of MXH10/Mo‐lpr/lpr, which develops a systemic swelling of LNs, and murine malignant fibrous histiocytoma‐like (KM‐Luc/GFP) cells or murine breast cancer (FM3A‐Luc) cells inoculated into the subiliac LN of mice to produce a tumor‐bearing LN model. The changes in INP during intranodal tumor progression and after treatment with cis‐dichlorodiammineplatinum(II) (CDDP) using an LDDS were measured. We found that tumor progression was associated with an increase in INP that occurred independently of LN volume changes. The elevation in INP was suppressed by CDDP treatment with the LDDS when intranodal tumor progression was significantly inhibited. These findings indicate that INP is a useful parameter for monitoring the therapeutic effect in patients with LN metastasis who have been given drugs using an LDDS, which will serve to manage cancer metastasis treatment and contribute to an improved quality of life for cancer patients.

Bovine lymph nodes as a source of Escherichia coli contamination of the meat

Ground beef contamination with Escherichia coli is usually a result of carcass faecal contamination during the slaughter process. Carcasses are contaminated when they come into contact with soiled hides or intestinal leakage content during dressing and the evisceration processes. A more recent and compelling hypothesis is that, when lymph nodes are present in manufacturing beef trimmings, they can be a potential source of Enterobacteriaceae contamination of ground beef. The aim of this study was to investigate the occurrence of E. coli in lymph nodes from beef carcasses used for ground meat production, in six slaughter plants situated in central Italy A total of 597 subiliac (precrural) lymph nodes were obtained from 597 cattle carcasses and screened for E. coli by culture. Furthermore, E. coli isolates (one per positive carcass) were tested for stx1, stx2 eaeA and hlyA genes that are commonly used to identify and characterise shiga toxin-producing E. coli (STEC). In addition, the E. coli isolates were profiled for antimicrobial susceptibility. A proportion of 34.2% (204/597) carcasses were positive for E. coli. PCR revealed that 29% (59/204) of E. coli possessed stx1 or stx2 which corresponded to 9.9% of the cattle sampled. Moreover, a combination of stx1 or stx2 and eaeA was found in in 4 isolates (2% among E. coli positive samples and 1% among cattle sampled) and a combination of stx1 or stx2 and eaeA and hly in 1 isolate (0.5% and 0.2%). More than 95% of isolates were susceptible to gentamicin, ceftriaxone, cyprofloxacin and cefotaxime while high rates of resistance were recorded for cephalotin, ampicillin, tetracycline, tripe sulfa and streptomycin. The multivariate analysis identified “age” as the factor most closely related to E. coli positivity (either generic E. coli or STEC) in bovine lymph nodes. In conclusion, subiliac lymph nodes represent a source of E. coli for ground beef. These results are of major importance for risk assessment and improving good manufacturing practices during animal slaughter and ground meat production.

Establishment and characterization of a porcine B cell lymphoma cell line

The lack of available, well characterized, established, domestic porcine cell lines hinders the advancement of porcine cellular immunology. A case of multicentric lymphoma was diagnosed in a market weight pig at the time of slaughter. Affected lymph nodes and spleen were collected and used for single cell isolation and analysis. Cell lines were established by 3 rounds of limiting dilution from splenic and subiliac lymph node lymphomas. Surface marker staining identified the cells as CD21+, CD79a+, CD20+, PAX5+, and CD3− and cells were grown and easily passaged in cell culture. Transcriptome analysis was carried out to further characterize these rapidly proliferating cells validating the initial cytometric findings, confirming their identity as B cell lymphomas, and suggesting that they arose from germinal center centroblasts with aberrant control of BCL6 expression. Functional analysis identified the cells as being involved in cancer, cell movement, cell survival, and apoptosis. These new porcine B cell lymphoma cell lines will be a valuable resource for more in-depth cellular investigations into the porcine immune system and cancer, as well as providing a potential tool for the growth of lymphotropic viruses of pigs and humans.

In vivo delivery of an exogenous molecule into murine T lymphocytes using a lymphatic drug delivery system combined with sonoporation

Physical delivery of exogenous molecules into lymphocytes is extremely challenging because conventional methods have notable limitations. Here, we evaluated the potential use of acoustic liposomes (ALs) and sonoporation to deliver exogenous molecules into lymphocytes within a lymph node (LN). MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice, which show systemic LN swelling, were used as the model system. After direct injection into the subiliac LN, a solution containing both ALs and TOTO-3 fluorophores (molecular weight: 1355) was able to reach the downstream proper axillary LN (PALN) via the lymphatic vessels (LVs). This led to the accumulation of a high concentration of TOTO-3 fluorophores and ALs in the lymphatic sinuses of the PALN, where a large number of lymphocytes were densely packed. Exposure of the PALN to >1.93 W/cm2 of 970-kHz ultrasound allowed the solution to extravasate into the parenchyma and reach the large number of lymphocytes in the sinuses. Flow cytometric analysis showed that TOTO-3 molecules were delivered into 0.49 ± 0.23% of CD8+7AAD− cytotoxic T lymphocytes. Furthermore, there was no evidence of tissue damage. Thus, direct administration of drugs into LVs combined with sonoporation can improve the delivery of exogenous molecules into primary lymphocytes. This technique could become a novel approach to immunotherapy.

Evaluation of two commercially-available Salmonella vaccines on Salmonella in the peripheral lymph nodes of experimentally-infected cattle.

Background:Salmonella is a common inhabitant of the ruminant gastrointestinal tract, where it often resides asymptomatically and may be shed into the feces. More recently it was discovered that Salmonella may be contained within the peripheral, non-mesenteric lymph nodes, where it is impervious to in-plant pathogen control interventions and may serve as a source of Salmonella-contamination of ground beef. Over the past 10 years considerable research effort has been expended at understanding how this pathogen gets to these lymph nodes, the duration of infection, and, most importantly, screening and developing potential intervention strategies that may be employed on farm prior to the animal being presented for slaughter.Methods:Utilizing an experimental model of Salmonella inoculation of bovine peripheral lymph nodes (PLNs), two pilot vaccine experiments were conducted to evaluate two Salmonella vaccines: Salmonella Newport Bacterial Extract (Experiment I) and Endovac-Bovi® (Experiment II) on preventing Salmonella acquisition by these nodes. In Experiment I, 4 months following the booster vaccination, 30 steers were inoculated with three Salmonella serotypes intradermally: Newport, Montevideo, and Anatum administered to the right legs, left legs, and to the caudal thorax and abdomen, respectively. Cattle were inoculated every other day over the course of five days (three total inoculation events) and 6 and 12 days following the final Salmonella inoculation, 16 and 14 head in each treatment were euthanized, respectively. In Experiment II, 12 head of Holstein steers were utilized. Seven days following the booster and weekly thereafter for 3 weeks (four total inoculation events), cattle were inoculated as above and euthanized 7 days following final inoculation. Right and left sub-iliac, popliteal and pre-scapular lymph nodes were collected in each experiment, weighed and cultured for Salmonella.Results:In Experiment I, no treatment differences were observed in Salmonella prevalence 6 days post-inoculation (necropsy 1). However, in vaccinated cattle at the second necropsy, a reduction (p = 0.05) in Salmonella prevalence was observed in the sub-iliac and pre-scapular lymph nodes as well as when all nodes were evaluated collectively (p = 0.04). In Experiment II, the vaccine reduced (p = 0.03) Salmonella prevalence in the right popliteal and tended (p = 0.09) to decrease prevalence in both popliteal lymph nodes.Conclusion:Under these experimental conditions, the data generated provide evidence of a partial vaccine effect on Salmonella within PLNs and indicate that further research may be warranted.

Toll-like Receptor 9 Activation Accelerates Inhibitor Formation in Response to Factor VIII

Inhibitor (neutralizing anti-drug antibody) formation against factor VIII (FVIII) is currently the most serious complication of FVIII replacement therapy for hemophilia A. The role of innate immune signals in this adaptive immune response is unclear. Lipopolysaccharide (LPS, the major component of the outer membrane in Gram-negative bacteria), which activates the innate immune sensor toll-like receptor 4 (TLR4), enhances inhibitor formation in hemophilia A mice. Intriguingly, earlier data suggested that activation of the endosomal DNA sensor TLR9 may actually suppress inhibitor formation. However, we demonstrated that a TLR9 agonist induced inhibitor formation against a factor IX transgene product in gene therapy for hemophilia B, which resulted from activation of monocyte-derived dendritic cells (moDCs) and enhancement of T follicular helper (Tfh) cell responses. Tfh cells drive germinal center (GC) formation and T cell help-dependent antibody formation. The aim of this study was to elucidate the role of TLR9 signaling in FVIII inhibitor formation in hemophilia A mice. Hemophilia A (F8e16-/-) B6/129 mice were co-injected IV (n=4) with FVIII (1.5 IU) and ODN-1826 (a class B CpG oligodeoxynucleotide, 50 µg), which is a TLR9 agonist, or injected on two consecutive days (n=3), first with ODN-1826 and then with FVIII on the next day. Control mice received FVIII only (n=5). Injections were performed once weekly for 4 weeks. Blood samples, spleens and subiliac (superficial inguinal) lymph nodes were collected for ELISA and Bethesda assays, and flow cytometry analysis. In our analysis, Tfh were defined as CD4+CXCR5+PD1+Bcl6- cells, while GC B cells were defined as CD19+GL7+CD95+. Tfh cell response and GC formation in the spleen were robustly enhanced by the TLR9 agonist compared to mice injected with FVIII only (2-fold for Tfh frequencies and 6-fold for GC B cells when co-injected; P&lt; 0.05). As a result, inhibitor titers increased &gt;400-fold (on average from 6.4 to 2746.5 BU/ml). In contrast, anti-FVIII IgG1 levels increased only 2.5-fold. The differences between mice injected on two consecutive days and the FVIII-only group were not statistically significant. A time-course experiment was also carried out to monitor progress of immune response to FVIII (1.5 IU administered once weekly) in the absence of TLR9 agonist over time: 4 (n=4), 6 (n=4) and 8 weeks (n=5) after the initial antigen challenge. Inhibitor titers continued to rise beyond the fourth week of antigen challenges, reaching 46-fold higher values at eighth week (from 3 BU/ml in the fourth week to 138 BU/ml in the eighth week, p&lt;0.05), while anti-FVIII IgG1 levels increased 2.5-fold (ns). These results suggest that the TLR9 agonist sped up the neutralizing response to FVIII, which otherwise progressed in the same manner (towards more neutralizing versus total anti-FVIII) but at a slower rate. Overall, TLR9 activation enhanced GC formation and accelerated neutralizing immune response to FVIII. Similar results were obtained in hemophilia A mice on a different strain background (F8e16-/- BALB/c). We propose that signaling via the innate immune receptor TLR9 leads to a more targeted immune response by reinforcing Tfh activation, likely through moDC activation, which results in enhanced germinal center formation and thus accelerated development of neutralizing antibodies. Disclosures No relevant conflicts of interest to declare.

Analysis of tumor vascularization in a mouse model of metastatic lung cancer

Therapies targeting tumor vasculature would improve the treatment of lung metastasis, although the early changes in vascular structure are incompletely understood. Here, we show that obstructive metastatic foci in lung arterioles decrease the pulmonary vascular network. To generate a mouse model of lung metastasis activation, luciferase-expressing tumor cells were inoculated into the subiliac lymph node (SiLN) of an MXH10/Mo-lpr/lpr mouse, and metastatic tumor cells in the lungs were activated by SiLN resection. Activation of metastases was monitored by in vivo bioluminescence imaging. Pulmonary blood vessel characteristics were analyzed using ex vivo micro-computed tomography. The enhanced permeability and retention (EPR) effect in neovasculature after tumor cell activation was evaluated from the accumulation of intravenously injected indocyanine green (ICG) liposomes. Metastatic foci in lung arterioles were investigated histologically. Micro-computed tomography revealed decreases in pulmonary blood vessel length, volume and number of branching nodes during the early stage of metastasis caused by metastatic foci. ICG liposome accumulation by the EPR effect was not detected. Histology identified metastatic foci in lung arterioles. The lack of an EPR effect after the formation of metastatic foci in lung arterioles makes conventional systemic chemotherapy ineffective for lung metastasis. Thus, alternative therapeutic methods of drug delivery are needed.

Morphological Aspects of the Superficial Lymph Nodes of Myrmecophaga tridactyla

Background: The giant anteater is a Xenarthra of the Myrmecophagidae family. It is classified in Brazil as vulnerable specie, however, in Rio de Janeiro and Rio Grande do Sul it is probably extinct. In an attempt to contribute to the preservation of the species, many injured animals are referred for treatment and rehabilitation, but the lack of information about morphology may hinder a more efficient clinical-surgical approach. The knowledge on the topography, dimensions and morphology of lymph nodes of a given species is essential for medical practice. The aim of this study was to identify and describe the general morphology of the superficial lymph nodes of Myrmecophaga tridactyla.Materials, Methods &amp; Results: Ten cadavers of adult Myrmecophaga tridactyla were used in this study. They were fixed and preserved with 10% formaldehyde solution and dissected throughout their length to locate their superficial lymph nodes. Lymph nodes of each center were removed for mesoscopic analysis. Measurements of width, length and thickness were obtained by a digital caliper. The superficial lymph nodes observed in the head were the mandibular and the intermandibular, while in the neck were identified superficial cervical and deep cervical lymph nodes. In the thoracic limbs, the axillary and the axillary accessory lymph nodes were present. In the pelvic limbs just one lymph node, the femoral, was observed, located in the thick subcutaneous fascia of the medial surface of the thigh. The main anatomical differences found in the lymph nodes of M. tridactyla are the absence of parotid, popliteal and subiliac lymph nodes and the presence of intermandibular lymph nodes and the developed femoral lymph nodes, which are unique in the pelvic limbs of this species. Moreover, some superficial lymph nodes were formed by several small lymph nodes, forming clusters, as observed in the proper and accessory axillary and femoral lymph nodes.Discussion: The lymph nodes of the head, neck and forelimbs of the M. tridactyla followed the pattern observed in domestic animals and rats, while the morphological pattern of the lymph nodes of the head and the hindlimb differed from that described in domestic animals. Different from the dogs and ruminants where a range of superficial lymph nodes can be normally palpated, such as the mandibular; retropharyngeal; superficial cervical; subiliac; popliteal; mammary and superficial inguinal, only the femoral lymph nodes are palpable in M. tridactyla. The main reasons for this are the greater thickness of the skin and the absence of certain classic lymphocytes in the anteater. The peculiar anatomical features of the head and neck of M. tridactyla may have induced changes in the lymph centers of these regions when compared to the morphologic pattern of the terrestrial mammals in general. The lymph drainage of the neck, forelimbs and trunk wall was considered to be similar to that of dog, since there is equivalence of lymph nodes between these two species. The lymphatic afferents of the abdominal wall, gluteal region and hindlimbs are probably directed to the large femoral lymph nodes, which differs from domestic animals, whose lymph are mainly drained to the subiliac, ischiatic and popliteal lymph nodes. The anatomical differences related to the number, size and topography of the lymph nodes observed in M. tridactyla may be relevant in the medical procedures of this species.