Abstract Colorectal cancer liver metastasis (CLM) is the most of common cause of death in patients with colorectal cancer (CRC) worldwide. There are various immune-suppressive and tumor-promoting mechanisms, which contribute to the unresponsiveness associated with current check point immunotherapy in CRC. Techniques such as Multiplex immunohistochemistry (mIHC) and automated image quantitation analysis, enable a deeper understanding of the diversity of the host’s anti-tumor immune response and could identify potential targets for immunotherapy. Herein, we present a 7-plex OPALTM protocol used to assess the composition and spatial distribution of T cell markers CD3, CD8, Foxp3 and CD103 and the epithelial to mesenchymal transition (EMT) markers alpha smooth muscle actin (α-SMA) and E-cadherin. The protocol has been manually optimized and validated in two independent cohorts of formalin-fixed, paraffin embedded CLM patient tissues (n=42) using well-established antibodies, single spectral library, negative controls and biological controls for corroborating staining pattern of α-SMA+ (sclerosed hemangioma samples) and T-cell infiltrates (benign liver). The accurate profiling of T- cell composition, location and phenotypic characterization could reveal important insights about the influence of T lymphocytes on prognosis after liver metastasectomy.

Most patients with colorectal cancer (CRC) develop metastases, predominantly in the liver (CLM). Targeted therapies are being investigated to improve current CLM treatments. This study tested the effectiveness of SAR131675, a selective VEGFR-3 tyrosine kinase inhibitor, to inhibit CLM in a murine model. Following intrasplenic induction of CLM, mice were treated daily with SAR131675. Tumor growth and immune infiltrates into tumor and liver tissues were assessed at 10-, 16- and 22-days post tumor induction by stereology, IHC and flow cytometry. SAR151675 treatment significantly reduced tumor burden and F4/80+ macrophages in the liver tissues. Analysis of immune cell infiltrates in liver showed tissue that at day 22, had the proportion of CD45+ leukocytes significantly reduced, particularly myeloid cells. Analysis of myeloid cells (CD11b+ CD45+) indicated that the proportion of F4/80− Ly6Clow was significantly reduced, including a predominate PD-L1+ subset, while CD3+ T cells increased, particularly CD8+ PD1+, reflected by an increase in the CD8+:CD4+ T cell ratio. In the tumor tissue SAR11675 treatment reduced the predominant population of F4/80+ Ly6Clo and increased CD4+ T cells. These results suggest that SAR131675 alters the immune composition within tumor and the surrounding liver in the later stages of development, resulting in a less immunosuppressive environment. This immunomodulation effect may contribute to the suppression of tumor growth.

(1) Liver regeneration following partial hepatectomy for colorectal liver metastasis (CRLM) has been linked to tumour recurrence. Inhibition of the renin–angiotensin system (RASi) attenuates CRLM growth in the non-regenerating liver. This study investigates whether RASi exerts an antitumour effect within the regenerating liver following partial hepatectomy for CRLM and examines RASi-induced changes in the tumour immune microenvironment; (2) CRLM in mice was induced via intrasplenic injection of mouse colorectal tumour cells, followed by splenectomy on Day 0. Mice were treated with RASi captopril (250 mg/kg/day), or saline (control) from Day 4 to Day 16 (endpoint) and underwent 70% partial hepatectomy on Day 7. Liver and tumour samples were characterised by flow cytometry and immunofluorescence; (3) captopril treatment reduced tumour burden in mice following partial hepatectomy (p < 0.01). Captopril treatment reduced populations of myeloid-derived suppressor cells (MDSCs) (CD11b+Ly6CHi p < 0.05, CD11b+Ly6CLo p < 0.01) and increased PD-1 expression on infiltrating hepatic tissue-resident memory (TRM)-like CD8+ (p < 0.001) and double-negative (CD4-CD8-; p < 0.001) T cells; (4) RASi reduced CRLM growth in the regenerating liver and altered immune cell composition by reducing populations of immunosuppressive MDSCs and boosting populations of PD-1+ hepatic TRMs. Thus, RASi should be explored as an adjunct therapy for patients undergoing partial hepatectomy for CRLM.

(1) Background: Recent clinical and experimental data suggests that the liver's regenerative response following partial hepatectomy can stimulate tumor recurrence in the liver remnant. The Wnt/β-catenin pathway plays important roles in both colorectal cancer carcinogenesis and liver regeneration. Studies have shown that the Wnt/β-catenin pathway regulates multiple renin-angiotensin system (RAS) genes, whilst RAS inhibition (RASi) reduces tumor burden and progression. This study explores whether RASi attenuates features of tumor progression in the regenerating liver post-hepatectomy by modulating Wnt/β-catenin signaling. (2) Methods: Male CBA mice underwent CRLM induction, followed one week later by 70% partial hepatectomy. Mice were treated daily with captopril, a RASi, at 250 mg/kg/day or vehicle control from experimental Day 4. Tumor and liver samples were analyzed for RAS and Wnt signaling markers using qRT-PCR and immunohistochemistry. (3) Results: Treatment with captopril reduced the expression of down-stream Wnt target genes, including a significant reduction in both c-myc and cyclin-D1, despite activating Wnt signaling. This was a tumor-specific response that was not elicited in corresponding liver samples. (4) Conclusions: We report for the first time decreased c-myc expression in colorectal tumors following RASi treatment in vivo. Decreased c-myc expression was accompanied by an attenuated invasive phenotype, despite increased Wnt signaling.

BACKGROUND AND OBJECTIVES:Neutrophils are emerging as a key player in periodontal pathogenesis. The surface expression of cellular markers enables functional phenotyping of neutrophils which have distinct roles in disease states. This study aimed to evaluate the effect of periodontal management on neutrophil phenotypes in peripheral blood in periodontitis patients over one year.MATERIALS AND METHODS:Peripheral blood and the periodontal parameters, mean probing depth and percentage of sites with bleeding on probing (%BOP), were collected from 40 healthy controls and 54 periodontitis patients at baseline and 3-, 6- and 12- months post-treatment. Flow cytometry was used to identify CD11b+ , CD16b+ , CD62L- and CD66b+ expression on neutrophils, neutrophil maturation stages as promyelocytes (CD11b- CD16b- ), metamyelocytes (CD11b+ CD16b- ) and mature neutrophils (CD11b+ CD16b+ ), and suppressive neutrophil phenotype as bands (CD16dim CD62Lbright ), normal neutrophils (CD16bright CD62Lbright ) and suppressive neutrophils (CD16bright CD62Ldim ).RESULTS:CD62L- expression decreased with treatment. No differences were observed in neutrophil maturation stages in health or disease upon treatment. Suppressive and normal neutrophils showed a reciprocal relationship, where suppressive neutrophils decreased with treatment and normal neutrophils increased with treatment. In addition, %BOP was associated with suppressive neutrophils.CONCLUSION:This study demonstrates that management of periodontitis significantly modifies distinct neutrophil phenotypes in peripheral blood. Suppressive neutrophils may play a role in the pathogenesis of periodontitis. However, their exact role is unclear and requires further investigation.

AIMS:T-cells are known to have a role in periodontitis, however, the effect of periodontal therapy on peripheral memory T-cells is unclear. This study evaluated variation in peripheral memory T-cells and red complex bacteria in sub-gingival plaque in patients undergoing periodontal management. METHODS:Peripheral blood mononuclear cells and sub-gingival plaque were collected from 54 periodontitis patients at baseline, 3-, 6- and 12-months post-therapy and 40 healthy controls. Periodontitis patients were divided into treatment outcome (TxO) groups based on prevalence of sites with probing depth ≥5 mm as good (<10% of sites), moderate (10-20%) or poor (>20%) at study conclusion. Naïve (TN -CCR7+ CD45RA+ ), central memory (TCM -CCR7+ CD45RA- ), effector memory (TEM -CCR7- CD45RA- ) and effector memory T-cells re-expressing CD45RA (TEMRA -CCR7- CD45RA+ ) were phenotyped using flow cytometry in CD4+ , CD8+ , CD4+ CD8+ and CD4- CD8- T-cells and red complex bacteria were quantified using qPCR. RESULTS:At baseline, periodontitis subjects had significantly greater mean probing depths and Porphyromonas gingivalis proportions, lower TN but higher CD4+ TCM , CD8+ TCM , CD4+ CD8+ TEM and CD4- CD8- TEM cell proportions compared to health. Periodontal therapy decreased mean probing depths, P. gingivalis proportions, TEM and CD4+ and CD8+ TCM cells, but increased TN and CD4+ and CD8+ TEMRA cells. The T-cell profile in the good TxO group showed therapy-related changes in CD4+ TEM , and CD8+ TN and TEM cells, whereas, no changes were observed in the poor TxO group. CONCLUSION:Management and the reduction in red complex bacteria were associated with changes in peripheral memory T-cells in periodontitis.

Background It is now recognized that many anticancer treatments positively modulate the antitumor immune response. Clinical and experimental studies have shown that inhibitors of the classical renin–angiotensin system (RAS) reduce tumor progression and are associated with better outcomes in patients with colorectal cancer. RAS components are expressed by most immune cells and adult hematopoietic cells, thus are potential targets for modulating tumor-infiltrating immune cells and can provide a mechanism of tumor control by the renin–angiotensin system inhibitors (RASi).Aim To investigate the effects of the RASi captopril on tumor T lymphocyte distribution in a mouse model of colorectal liver metastases.Methods Liver metastases were established in a mouse model using an autologous colorectal cancer cell line. RASi (captopril 750 mg/kg) or carrier (saline) was administered to the mice daily via intraperitoneal injection, from day 1 post-tumor induction to endpoint (day 15 or 21 post-tumor induction). At the endpoint, tumor growth was determined, and lymphocyte infiltration and composition in the tumor and liver tissues were analyzed by flow cytometry and immunohistochemistry (IHC).Results Captopril significantly decreased tumor viability and impaired metastatic growth. Analysis of infiltrating T cells into liver parenchyma and tumor tissues by IHC and flow cytometry showed that captopril significantly increased the infiltration of CD3+ T cells into both tissues at day 15 following tumor induction. Phenotypical analysis of CD45+ CD3+ T cells indicated that the major contributing phenotype to this influx is a CD4 and CD8 double-negative T cell (DNT) subtype, while CD4+ T cells decreased and CD8+ T cells remained unchanged. Captopril treatment also increased the expression of checkpoint receptor PD-1 on CD8+and DNT subsets .Conclusion Captopril treatment modulates the immune response by increasing the infiltration and altering the phenotypical composition of T lymphocytes and may be a contributing mechanism for tumor control.

AIM:Periodontitis has been associated with other systemic diseases with underlying inflammation responsible for the shared link. This study evaluated longitudinal variation in peripheral T helper cells in periodontitis patients undergoing management over 1 year. MATERIALS AND METHODS:Periodontal parameters and peripheral blood mononuclear cells (PBMCs) were collected from 54 periodontitis patients at baseline, and 3-, 6- and 12-months post-treatment and 40 healthy controls. IFN-γ+ , IL-4+ , IL-17+ and Foxp3+ and their double-positive expression were identified in CD4+ and TCRαβ+ cells using flow cytometry. PBMCs were incubated with P. gingivalis, and IFN-γ, IL-4, IL-17 and IL-10 in cell supernatant were measured by ELISA. Cells and cytokines were also assessed based on clinical response to treatment where good (<10% of sites), moderate (10-20%) and poor (>20%) treatment outcome (TxO) groups had probing depths of ≥5 mm at study conclusion. RESULTS:IFN-γ+ cells were lower at baseline, and 3- and 6-months compared to health, whereas Foxp3+ cells were increased at 12-months compared to all preceding timepoints and health. The good TxO group showed treatment-related variation in IFN-γ+ and Foxp3+ cells, whereas the poor TxO group did not. IFN-γ and IL-17 cytokine expression in cell supernatants was significantly lower at baseline compared to health, and IFN-γ and IL-10 showed treatment-related decrease. CONCLUSION:This study suggests that IFN-γ+ and Foxp3+ cells may have a role in the systemic compartment in periodontitis. Periodontal management has local and systemic effects, and thus, assessment and management of periodontitis should form an integral part of overall systemic health.

Periodontitis is a chronic inflammatory disease with a complex underlying immunopathology. Cytokines, as molecular mediators of inflammation, play a role in all stages of disease progression. T helper 17 (Th17) cells are thought to play a role in periodontitis. Th17 cell development and maintenance requires a pro-inflammatory cytokine milieu, with many of the cytokines implicated in the pathogenesis of periodontitis. Serum and saliva are easily accessible biofluids which can represent the systemic and local environment to promote the development of Th17 cells. Here we review human clinical studies that investigate IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, sCD40L and TNF-α in serum and saliva in periodontitis. We highlight their putative role in the pathogenesis of periodontitis and place them within a wider context of animal and other clinical studies.

Purpose: Extended radical resection (ERR) represents the only potentially curative option for patients with advanced pelvic malignancy.Tumours involving the pelvic sidewall, and in particular the sciatic nerve, have traditionally been considered inoperable.Small studies have demonstrated encouraging functional outcomes after proximal lower limb sarcoma resection involving the sciatic nerve.This study aimed to report survival, functional and quality of life (QoL) outcomes after ERR with en bloc sciatic or femoral nerve excision.Methodology: Consecutive patients who underwent ERR for advanced pelvic malignancy with en bloc resection of the sciatic or femoral nerves were included.Results: 713 patients underwent ERR, of which 68 (9.5%) required resection of a major nerve.Complete sciatic, partial sciatic and complete femoral nerve resection was required in 26 (38%), 38 (56%) and 4 patients (6%), respectively.27 patients (40%) experienced a major postoperative complication.R0 resection rate was 65%.In patients with colorectal cancer, overall and local recurrence-free 5-year survival rates were 55% and 76% after R0 resection.22 (96%) and 25 (92%) patients could mobilize independently or with an aid after complete and partial sciatic nerve resection, respectively.At 6 months after surgery, physical QoL was significantly lower than baseline (P = 0.041), but returned to baseline by 12 months (P = 0.163).Conclusion: Excision of the sciatic and femoral nerve can be performed during ERR with morbidity and survival outcomes comparable to existing literature where nerve resection is not performed.Physical QoL may be temporarily reduced, but returns to baseline by 12 months.Functional outcomes are better than anticipated.

Aim: T helper (Th)17 cells are implicated in the pathogenesis of periodontitis. This study investigated the effect of periodontal management on fifteen Th17-related cytokines in serum and saliva in periodontitis patients. Materials and methods: Periodontal parameters, serum and saliva were collected from 40 healthy controls and 54 periodontitis subjects before treatment, and 3-, 6- and 12-months post-treatment. Cytokine concentrations of IL-1 beta IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-gamma, sCD40L and TNF-alpha were determined by Luminex assay. Results: IL-1 beta, IL-6, sCD40L and TNF-alpha in serum, and IL-1 beta, IL-6, IL-25 and IL-31 in saliva were significantly higher at baseline compared to health and decreased with treatment. In contrast, serum IL-31 was significantly lower at baseline compared to health and increased with treatment. In addition, salivary IL-10, IL-17A, IL-17F, IL-23, IL-33, IFN-gamma and TNF-alpha also displayed treatment-related reduction. Correlation networks showed that cytokines in saliva displayed a higher number of correlations compared to serum in periodontitis. Conclusion: Treatment generally decreased cytokine concentrations except for serum IL-31 which showed a treatment-related increase. Serum cytokine concentrations may not be reflective of salivary cytokines. Saliva may be a better medium for cytokine detection compared to serum. Serum IL-31 and salivary IL-1 beta, IL-6, IL-10 and TNF-alpha were significant predictors for mean probing depth and may be potential biomarkers of interest in the pathogenesis of periodontitis.

Immunotherapies have not yielded significant clinical benefits for pancreatic ductal adenocarcinoma (PDA) because of the existence of an immunosuppressive tumour microenvironment (TME) characterized by a desmoplastic stroma containing infiltrated immune cells and activated pancreatic stellate cells (PSCs). This study aims to investigate the involvement of PAK1 in anti-tumour immunity. In PDA patients, low PAK1 expression, low activation of PSC and high CD8+ T cell/PAK1 ratios correlated with longer overall survival. In a murine PDA model, PAK1 knockout increased intra-tumoral CD4+ and CD8+ T cells, inhibited PSCs activation and extended survival. Inhibition of PAK1 reduced PSC-stimulated PDA cell proliferation and migration, blocked PSC-mediated protection of PDA cells from killing by cytotoxic lymphocytes and decreased intrinsic and PSC-stimulated PD-L1 expression in PDA cells, which further sensitized PDA cells to cytotoxic lymphocytes. Inhibition of PAK1 stimulates anti-tumour immunity by increasing intra-tumoral CD4+ and CD8+ T cells, and by sensitizing PDA cells to killing by cytotoxic lymphocytes via down-regulation of intrinsic and PSC-stimulated PD-L1 expression. PAK1 inhibitors, especially in combination with immune checkpoint inhibitors may result in improved efficacy of immunotherapy of PDA.

Interleukin (IL)‐36 cytokines are important regulators of mucosal homeostasis and inflammation. We previously established that oral epithelial cells strongly upregulate IL‐36γ expression in response to the bacterial pathogen Porphyromonas gingivalis. Here, we have established that IL‐36γ stimulates the expression of the IL‐12 cytokine family members, IL‐23p19 and Epstein‐Barr Virus‐Induced Gene 3 (EBI3), by oral epithelial cells; their expression was also selectively stimulated by IL‐36α. Notably, IL‐23p19 and EBI3 expression was not stimulated by P. gingivalis, thus suggesting that their expression by the oral epithelium in response to P. gingivalis is likely to be mediated in an autocrine manner by IL‐36γ. The IL‐36γ‐inducible expression of IL‐23p19 and EBI3 was found to be diametrically regulated by the mitogen‐activated protein kinase/extracellular signal regulated kinase (MEK)‐extracellular signal‐regulated kinase 1/2 (ERK1/2) pathway, whereby the activation of MEK‐ERK signaling likely functions as a negative feedback mechanism to limit EBI3 expression. Furthermore, epidermal growth factor receptor (EGFR) signaling, which is important for mucosal homeostasis, was demonstrated to modulate, in a MEK‐ERK‐dependent manner, the stimulation of IL‐23p19 and EBI3 expression by IL‐36γ. IL‐23p19 and EBI3 have recently been shown to heterodimerize to form the novel cytokine IL‐39 and promote neutrophil expansion. EBI3 has been shown to also have IL‐12 cytokine family independent functions (e.g. mediating IL‐6 trans‐signaling). Thus, this study not only advances our understanding of how IL‐36 cytokines may control mucosal inflammation, but also establishes EGFR signaling as a potentially important modulator of IL‐36 cytokine function.

Interleukin (IL)-36 cytokines are important regulators of mucosal homeostasis and inflammation. We previously established that oral epithelial cells strongly upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis . Here, we have established that IL-36γ stimulates the expression of the IL-12 cytokine family members, IL-23p19 and Epstein-Barr Virus-Induced Gene 3 ( EBI3 ), by oral epithelial cells; their expression was also selectively stimulated by IL-36α. Notably, IL-23p19 and EBI3 expression was not stimulated by P. gingivalis , thus suggesting that their expression by the oral epithelium in response to P. gingivalis is likely to be mediated in an autocrine manner by IL-36γ. The IL-36γ-inducible expression of IL-23p19 and EBI3 was found to be diametrically regulated by the mitogen-activated protein kinase/extracellular signal regulated kinase (MEK)-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereby the activation of MEK-ERK signaling likely functions as a negative feedback mechanism to limit EBI3 expression. Furthermore, epidermal growth factor receptor (EGFR) signaling, which is important for mucosal homeostasis, was demonstrated to modulate, in a MEK-ERK-dependent manner, the stimulation of IL-23p19 and EBI3 expression by IL-36γ. IL-23p19 and EBI3 have recently been shown to heterodimerize to form the novel cytokine IL-39 and promote neutrophil expansion. EBI3 has been shown to also have IL-12 cytokine family independent functions (e.g. mediating IL-6 trans -signaling). Thus, this study not only advances our understanding of how IL-36 cytokines may control mucosal inflammation, but also establishes EGFR signaling as a potentially important modulator of IL-36 cytokine function.

Renin-angiotensin system inhibitors (RASi) have shown potential anti-tumor effects that may have a significant impact in cancer therapy. The components of the renin-angiotensin system (RAS) including both, conventional and alternative axis, appear to have contradictory effects on tumor biology. The mechanisms by which RASi impair tumor growth extend beyond their function of modulating tumor vasculature. The major focus of this review is to analyze other mechanisms by which RASi reprogram the tumor immune microenvironment. These involve impairing hypoxia and acidosis within the tumor stroma, regulating inflammatory signaling pathways and oxidative stress, modulating the function of the non-cellular components and immune cells, and regulating the cross-talk between kalli krein kinin system and RAS.

Objective: Family environments have an effect on physical health during adolescence, and a possible underlying mechanism is inflammation. However, little is known about the association between observed parenting behaviors and immune system functioning. The current study examined whether positive and negative emotional parental behaviors observed during family interactions were associated with inflammation in adolescents. Method: Sixty-one parent-adolescent dyads (37 male adolescents, 60.6%; 15 male parents, 24.6%) were observed during 2 laboratory-based interaction tasks designed to elicit positive and conflictual emotional behaviors, respectively. Frequency of aggressive and positive parental behavior was coded. Adolescents were followed up approximately 2.5 years later and salivary concentrations of the inflammatory biomarker C-reactive protein (sCRP) were measured. Results: Controlling for BMI and depressive symptoms, lower sCRP was associated both with greater frequency of positive parental behaviors, t = −3.087, p = .003 and less frequency of aggressive parental behavior (t = 2.087, p = .041) in the conflictual task. Trend associations between positive behavior during the positive task and lower sCRP were also found. Conclusions: This is the first study to show that observed positive parenting is associated with lower levels of inflammation in adolescents.

OBJECTIVE:Temperament has associations with later physical health outcomes, yet there is a dearth of research exploring the connection between temperament and mechanisms that have known associations with these health outcomes. Recent research has delineated a connection between personality and inflammation during adulthood, but this association has not yet been studied in adolescent samples. DESIGN:We investigated whether stable adolescent temperament (averaged over two years), specifically effortful control and negative emotionality, provided a more robust prediction of inflammation as measured by salivary C-reactive protein (sCRP), than depressive symptoms. METHODS:Temperament and depressive symptoms were measured in a sample of sixty-three adolescents (37 males) when they were approximately 12 years old (mean age = 12.30, SD = 0.69) and again when they were approximately 14 years old (mean age = 14.84, SD = 0.49). Levels of sCRP were determined approximately 7 months later (mean = 6.77, SD = 2.99) when participants were approximately 15 years old (mean age = 15.49, SD = 0.49). RESULTS:Regression analyses revealed that effortful control (EC) was significantly associated with lower sCRP levels, while higher negative emotionality (NE) was significantly associated with higher sCRP levels. Furthermore, these associations were larger than those for depressive symptoms and were differentially impacted by the addition of covariates. Implications for the role of stable risk and protective factors in inflammatory processes are discussed. CONCLUSIONS:These findings are the first to show associations between adolescent temperament and inflammation. Furthermore, these findings extend previous personality research to temperamental research in a younger sample of adolescents. Statement of contribution What is already known? There is a large extant literature on the association between depressive symptoms and inflammation. There is a smaller extant literature on the association between personality and inflammation. No studies have examined how adolescent temperament traits may relate to inflammation. What does this study add? Longitudinal data collection over the course of 3 years in an adolescent sample. Addresses the question of whether temperament factors relate to inflammation. Temperament provides a more robust predictor of later inflammation than depressive symptoms.

We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy.