Development and assessment of a high-throughput biofilm and biomass testing platform.

OBJECTIVE: To develop and evaluate a simple platform technology for developing static biofilms in a 96-well microtitre plate for various downstream applications. The technology allows monitoring of growth rate, biofilm formation and quantifying biofilm biomass by using crystal violet (CV) and safranin O (SO) staining over seven-day time periods for pathogens including clinical isolates most commonly associated with hard-to-treat wound infections. METHOD: A total of 157 bacteria including Acinetobacter, Enterobacter, Klebsiella, Pseudomonas and Staphylococcus spp. were used in the study. Bacterial growth was measured at 600nm optical density (OD). Biofilm formation was monitored and assessed quantitatively with CV at 570nm and SO staining at 492nm for one-, two-, three- and seven-day incubation periods. RESULTS: Bacterial growth rate and static biofilm biomass in the 96-well plates varied for various strains tested. Both CV and SO staining showed similar results in the biomass, with SO assay displaying more reproducible data throughout the study. Most of the strains were metabolically active even at the seven-day incubation period. Microbial adherences of all bacterial strains on the plastic surface was assessed with CV staining: 28 Acinetobacter, 17 Staphylococcus, 12 Pseudomonas and four Enterobacter strains were strong biofilm producers. Moderate biofilm-producing strains included 27 Staphylococcus, 14 Acinetobacter, eight Pseudomonas and three Enterobacter. Weak biofilm-producing strains included: 33 Staphylococcus, six Enterobacter, two Pseudomonas and one Acinetobacter. Only one Pseudomonas aeruginosa strain did not develop biofilm. CONCLUSION: Our results demonstrate the feasibility of using 96-well microtitre plates as a high-throughput platform for quantitative measurement and assessment of biofilm development over time. Studying microbial adherence or biofilm biomass generated on various surfaces using a high-throughput system could provide valuable information for in vitro testing and developing therapeutics for biofilm infections. Employing the biofilm testing platform described in this study makes it possible to simultaneously develop different biofilms formed by specific pathogens, and study potential association between the quantity of bacterial biomass and strength of a biofilm formed by specific wound pathogens. In addition, the described testing approach could provide an optimal model for standardised and high-throughput screening of candidate antibiofilm therapeutics.

Medical applications of cold atmospheric plasma: state of the science.

Cold atmospheric plasmas (CAP) have been used in multiple medical fields and have become a promising medical technology. CAP-generating devices are safe and easy to operate and can now be manufactured at a low cost due to advancements in electronics and microchips. A primary application of CAP is as a broad-spectrum antimicrobial technology. With the high incidence of infections caused by drug-resistant microorganisms, a non-antibiotic based treatment modality such as CAP holds great therapeutic promise, particularly in the wound care field. In addition to its antimicrobial properties, CAP treatment enhances wound healing by increasing cutaneous microcirculation, monocyte stimulation, and keratinocyte proliferation. CAP has been used by dentists for disinfection of teeth, enhancing gingival fibroblast activity, and even teeth whitening. CAP can combat tumour growth by increasing the efficacy of antitumour therapeutic agents, reactivating apoptotic pathways, or down-regulating growth-related gene sites. Most of the health-care related research on CAP has occurred in the past 15 years; the field is relatively young and needs additional research, as well as confirmation of the existing supporting literature. The purpose of this report is to provide the reader with an overview of the therapeutic application of the cold plasma technology.

Antiviral Efficacy Testing of a Rechargeable Textile.

INTRODUCTION: In light of the COVID-19 (Coronovirus Disease 2019) pandemic, the use of personal protective equipment has become essential to reduce viral transmission and maintain public health. Viruses, particularly human coronavirus and influenza, pose significant challenges because of their various transmission routes. UMF Corporation's innovation, Micrillon, aims to address these challenges by creating durable, antiviral technology for textiles without harmful chemicals, reducing viral transmission risks. MATERIALS AND METHODS: The study followed ISO Standard 18184:2019, testing Micrillon textiles against Human Coronavirus OC43 and H1N1 Influenza A virus using MDCK and HCT-8 cell lines. Cell propagation, viral application, TCID50 (Median Tissue Culture Infectious Dose) testing, and maintenance protocols were rigorously implemented to assess antiviral efficacy. RESULTS: Micrillon gloves, fabrics, and fibers exhibited high antiviral efficacy against both viruses across various contact times. Gloves demonstrated exceptional antiviral activity against H1N1 (99.88%) and OC43 (99.67%) at 120 minutes. Rolled fabrics showed strong efficacy against H1N1 (99.42% at 30 minutes) and OC43 (>97%) at all time points. Bundled fibers displayed substantial efficacy against H1N1 (99.17% at 120 minutes) and OC43 (>98%) at all time points. CONCLUSIONS: The study demonstrates that Micrillon technology effectively inhibits viral activity, particularly in gloves, fabrics, and fibers. The innovation not only shows high antiviral efficacy against both Human Coronavirus and Influenza but also promises a reusable, sustainable solution, mitigating environmental impact and reducing the use of harmful chemicals in personal protective equipment. The technology holds promise for widespread use in health care and hospitality, offering a layer of protection while being environmentally conscious. Further development of such technologies can significantly reduce infection risks while minimizing environmental harm.

Differential expression of colon cancer associated transcript1 (CCAT1) along the colonic adenoma-carcinoma sequence.

BACKGROUND: The transition from normal epithelium to adenoma and, to invasive carcinoma in the human colon is associated with acquired molecular events taking 5-10 years for malignant transformation. We discovered CCAT1, a non-coding RNA over-expressed in colon cancer (CC), but not in normal tissues, thereby making it a potential disease-specific biomarker. We aimed to define and validate CCAT1 as a CC-specific biomarker, and to study CCAT1 expression across the adenoma-carcinoma sequence of CC tumorigenesis. METHODS: Tissue samples were obtained from patients undergoing resection for colonic adenoma(s) or carcinoma. Normal colonic tissue (n = 10), adenomatous polyps (n = 18), primary tumor tissue (n = 22), normal mucosa adjacent to primary tumor (n = 16), and lymph node(s) (n = 20), liver (n = 8), and peritoneal metastases (n = 19) were studied. RNA was extracted from all tissue samples, and CCAT1 expression was analyzed using quantitative real time-PCR (qRT-PCR) with confirmatory in-situ hybridization (ISH). RESULTS: Borderline expression of CCAT1 was identified in normal tissue obtained from patients with benign conditions [mean Relative Quantity (RQ) = 5.9]. Significant relative CCAT1 up-regulation was observed in adenomatous polyps (RQ = 178.6 ± 157.0; p = 0.0012); primary tumor tissue (RQ = 64.9 ± 56.9; p = 0.0048); normal mucosa adjacent to primary tumor (RQ = 17.7 ± 21.5; p = 0.09); lymph node, liver and peritoneal metastases (RQ = 11,414.5 ± 12,672.9; 119.2 ± 138.9; 816.3 ± 2,736.1; p = 0.0001, respectively). qRT-PCR results were confirmed by ISH, demonstrating significant correlation between CCAT1 up-regulation measured using these two methods. CONCLUSION: CCAT1 is up-regulated across the colon adenoma-carcinoma sequence. This up-regulation is evident in pre-malignant conditions and through all disease stages, including advanced metastatic disease suggesting a role in both tumorigenesis and the metastatic process.

CD8 knockout mice are protected from challenge by vaccination with WR201, a live attenuated mutant of Brucella melitensis.

CD8+ T cells have been reported to play an important role in defense against B. abortus infection in mouse models. In the present report, we use CD8 knockout mice to further elucidate the role of these cells in protection from B. melitensis infection. Mice were immunized orally by administration of B. melitensis WR201, a purine auxotrophic attenuated vaccine strain, then challenged intranasally with B. melitensis 16M. In some experiments, persistence of WR201 in the spleens of CD8 knockout mice was slightly longer than that in the spleens of normal mice. However, development of anti-LPS serum antibody, antigen-induced production of γ-interferon (IFN-γ) by immune splenic lymphocytes, protection against intranasal challenge, and recovery of nonimmunized animals from intranasal challenge were similar between normal and knockout animals. Further, primary Brucella infection was not exacerbated in perforin knockout and Fas-deficient mice and these animals' anti-Brucella immune responses were indistinguishable from those of normal mice. These results indicate that CD8+ T cells do not play an essential role as either cytotoxic cells or IFN-γ producers, yet they do participate in a specific immune response to immunization and challenge in this murine model of B. melitensis infection.

Sequential real-time PCR assays applied to identification of genomic signatures in formalin-fixed paraffin-embedded tissues: a case report about brucella-induced osteomyelitis.

Brucellosis is a zoonotic infection transmitted from animals to human by ingestion of infected food products, direct contact with an infected animal, or inhalation of aerosols. Brucella infection-induced osteomyelitis may present only with nonspecific clinical and radiographic findings, mild elevations in serum inflammatory markers, as well as nonspecific histological changes. We studied a case of an Iraqi war veteran with multifocal vertebral body and left iliac bone lesions on radio nucleotide scans and magnetic resonance imaging, clinically suspected osteomyelitis possibly because of Brucella. Although histomorphological findings were nonspecific, consisting of chronic inflammatory cell infiltrate and reactive fibrosis, tissue gram and silver impregnation stains of bone biopsies were informative, revealing gram-negative coccobacilli consistent in size with Brucella species. Total nucleic acids were extracted from formalin-fixed paraffin-embedded tissues and amplified by sequential real-time polymerase chain reaction, targeting genes coding (1) outer membrane protein (omp-31) of Brucella species and (2) insertion sequence (IS711) of Brucella abortus (b-abt). Polymerase chain reaction results confirmed B. abortus as the causative pathogens for presumed diagnosis of Brucella osteomyelitis.

Morpholino oligonucleotides do not participate perfectly in standard Watson-Crick complexes with RNA.

RNase P from E. coli will cleave a RNA at a site designated in a complex with an external guide sequence (EGS). The location of the site is determined by the Watson-Crick complementary sequence that can be formed between the RNA and the EGS. Morpholino oligonucleotides (PMOs) that have the same base sequences as any particular EGS will not direct cleavage by RNase P of the target RNA at the expected site in three mRNAs. Instead, cleavage occurs at a secondary site that does not correspond exactly to the expected Watson-Crick sequence in the PMO. This cleavage in the mRNA for a drug resistance gene, CAT mRNA, is at least second order in the concentration of the PMOs, but the mechanism is not understood yet and might be more complicated than a simple second-order reaction. EGSs and PMOs inhibit the reactions of each other effectively in a competitive fashion. A basic peptide attached to the PMO (PPMO) is more effective because of its binding properties to the mRNA as a substrate. However, a PMO is just as efficient as a PPMO on a mRNA that is mutated so that the canonical W-C site has been altered. The altered mRNA is not recognizable by effective extensive W-C pairing to an EGS or PMO. The complex of a PMO on a mutated mRNA as a substrate shows that the dimensions of the modified oligonucleotide cannot be the same as a naked piece of single-stranded RNA.

Inactivation of expression of several genes in a variety of bacterial species by EGS technology.

The expression of gene products in bacteria can be inhibited by the use of RNA external guide sequences (EGSs) that hybridize to a target mRNA. Endogenous RNase P cleaves the mRNA in the complex, making it inactive. EGSs participate in this biochemical reaction as the data presented here show. They promote mRNA cleavage at the expected site and sometimes at other secondary sites. Higher-order structure must affect these reactions if the cleavage does not occur at the defined site, which has been determined by techniques based on their ability to find sites that are accessible to the EGS oligonucleotides. Sites defined by a random EGS technique occur as expected. Oligonucleotides made up primarily of defined or random nucleotides are extremely useful in inhibiting expression of the gyrA and rnpA genes from several different bacteria or the cat gene that determines resistance to chloramphenicol in Escherichia coli. An EGS made up of a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) does not cleave at the same site as an unmodified RNA EGS for reasons that are only partly understood. However, PPMO-EGSs are useful in inhibiting the expression of targeted genes from Gram-negative and Gram-positive organisms during ordinary growth in broth and may provide a basis for broad-spectrum antibiotics.

Development of hydrolysis probe-based real-time PCR for identification of virulent gene targets of Burkholderia pseudomallei and B. mallei--a retrospective study on archival cases of service members with melioidosis and glanders.

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria responsible for melioidosis and glanders, respectively. Our laboratory developed hydrolysis probe-based real-time polymerase chain reaction assays targeting type three secretion system (TTS) and transposase family protein (TFP) of B. pseudomallei and B. malli, respectively. The assays were validated for target specificity, amplification sensitivity, and reproducibility. A bacterial DNA panel, composed of B. pseudomallei (13 strains), B. mallei (11 strains), Burkholderia species close neighbors (5 strains), and other bacterial species (17 strains), was prepared for specificity testing. Reference DNAs from B. pseudomallei and B. mallei bacterial cultures were used as controls for amplification, limit of detection, and reproducibility testing. The two TaqMan assays, Bp-TTS 1 and Bm-TFP, were optimized and applied in a retrospective study of archived cases from the Armed Forces Institute of Pathology. We tested 10 formalin-fixed paraffin-embedded blocks originally from autopsy specimens of patients who died of melioidosis or glanders during or after overseas tours in 1960s. Polymerase chain reaction results confirmed that DNA samples from formalin-fixed paraffin-embedded blocks of eight patients with melioidosis were positive for Bp-TTS 1 target and two patients with glanders were positive for Bm-TFP target.

Rapidly Self-Sterilizing PPE Capable of Destroying 100% of Microbes in 30-60 Seconds.

The continued proliferation of superbugs in hospitals and the coronavirus disease 2019 (COVID-19) has created an acute worldwide demand for sustained broadband pathogen suppression in households, hospitals, and public spaces. In response, we have created a highly active, self-sterilizing copper configuration capable of inactivating a wide range of bacteria and viruses in 30-60 seconds. The highly active material destroys pathogens faster than any conventional copper configuration and acts as quickly as alcohol wipes and hand sanitizers. Unlike the latter, our copper material does not release volatile compounds or leave harmful chemical residues and maintains its antimicrobial efficacy over sustained use; it is shelf stable for years. We have performed rigorous testing in accordance with guidelines from U.S. regulatory agencies and believe that the material could offer broad spectrum, non-selective defense against most microbes via integration into masks, protective equipment, and various forms of surface coatings.

Inhibition of expression of virulence genes of Yersinia pestis in Escherichia coli by external guide sequences and RNase P.

External guide sequences (EGSs) targeting virulence genes from Yersinia pestis were designed and tested in vitro and in vivo in Escherichia coli. Linear EGSs and M1 RNA-linked EGSs were designed for the yscN and yscS genes that are involved in type III secretion in Y. pestis. RNase P from E. coli cleaves the messages of yscN and yscS in vitro with the cognate EGSs, and the expression of the EGSs resulted in the reduction of the levels of these messages of the virulence genes when those genes were expressed in E. coli.

A Novel Peptide-Based Antimicrobial Wound Treatment is Effective Against Biofilms of Multi-Drug Resistant Wound Pathogens.

Wound infections are a common complication of combat-related injuries that significantly increase morbidity and mortality. Multi-drug resistant (MDR) organisms and their associated biofilms play a significant role in the pathogenicity and chronicity of wound infections. A critical barrier to progress in the treatment of traumatic wounds is the need for broad spectrum antimicrobials that are effective against biofilms and compatible with topical delivery. In this study, we present the in vitro efficacy of two de novo designed cationic, antimicrobial peptides and related topical formulations against single species and polymicrobial biofilms of MDR bacteria. Minimum biofilm eradication concentrations for peptides ranged from 0.7 µM for Staphylococcus aureus to 13.2 µM for Pseudomonas aeruginosa. Varying pH did not adversely impact peptide activity, however, in the presence of albumin, minimum biofilm eradication concentrations generally increased. When formulated into gels or dressings, both peptides eradicated mono- and polymicrobial biofilms of MDR pathogens. The biocompatibility index (BI) was found to be greater than one for both ASP-1 and ASP-2, with a slightly greater (more favorable) BI for ASP-2. The BIs for both peptides were greater than BIs previously reported for commonly used topical antimicrobial agents. The antimicrobial peptides and related formulations presented provide a promising platform for treatment of wound biofilms to improve outcomes for those injured in combat.

A Prospective Randomized Controlled Two-Arm Clinical Study Evaluating the Efficacy of a Bioelectric Dressing System for Blister Management in US Army Ranger Recruits.

This study focused on a clinically relevant healthcare problem in the military: acute soft tissue wounds, or blisters. The trial was a prospective, controlled, randomized two-arm study evaluating the efficacy of a bioelectric dressing, Procellera®, applied topically two to three times per week for 2 weeks to blisters developed in Ranger trainees during training at Fort Benning, Georgia. A total of 80 US Army Ranger recruits with blister wounds below the knee were randomly assigned to one of two treatment groups (n = 40/group). The primary goal was to assess the clinical efficacy (rate of healing) of administered Procellera in conjunction with the standard-of-care (SOC) treatment, moleskin and Tegaderm ®, on the healing rate of blisters compared with the SOC treatment alone. The secondary end points for efficacy were the quantities of wound fluid biomarkers and bacterial bioburden. The tertiary end point was assessment of pain in the treatment group compared with that of the control group during the 2-week study. The results showed no statistical difference between the SOC and SOC+Procellera groups in wound healing and pain. Wound fluid was reported for 24 participants (64.9%) in the SOC group and 21 participants (56.8%) in SOC+Procellera group at the baseline measurement (ρ = .475); however, the wounds were devoid of fluid on follow-up visits. The mild nature of the wounds in this study was apparent by the low pain scores at the beginning of the study, which disappeared by the follow-up visits. The average wound sizes were 2.2cm2 and 1.5cm2 for the SOC and SOC+Procellera groups, respectively. This trial protocol should be conducted on open softtissue wounds in severe heat. To our knowledge, this is the first clinical study conducted within the US Army Rangers training doctrine.

An Overview of the Efficacy of a Next Generation Electroceutical Wound Care Device.

Novel approaches including nonpharmacological methodologies for prevention and control of microbial pathogens and emerging antibiotic resistance are urgently needed. Procellera is a wound care device consisting of a matrix of alternating silver (Ag) and zinc (Zn) dots held in position on a polyester substrate with a biocompatible binder. This electroceutical medical device is capable of generating a direct current voltage (0.5-0.9 Volts). Wound dressings containing metals such as Ag and/or Zn as active ingredients are being used for control of colonized and infected wounds. Reports on the presence of electric potential field across epithelium and wound current on wounding have shown that wound healing is enhanced in the presence of an external electrical field. However, majority of the electrical devices require an external power source for delivering pulsed or continuous electric power at the wound site. A microelectric potential-generating system without an external power source is an ideal treatment modality for application in both clinical and field settings. The research presented herein describes efficacy evaluation of a wireless bioelectric dressing against both planktonic and biofilm forms of wound pathogens including multidrug resistant organisms.

Differential Diagnosis of Candida Species With Real-Time Polymerase Chain Reaction and Melting Temperature Analyses (RTPCR-MTA).

Genus Candida covers more than 50 species, half of which can cause infections in humans. Some of the Candida species exhibit drug resistance; therefore, there is an urgent need for rapid and accurate differentiation for rendering appropriate and effective management. Here, we report a new methodology employing real-time polymerase chain reaction (RTPCR) and melting temperature analyses (MTA) procedures. Fungal ribosomal internal transcribed spacer 2 (ITS2) has been confirmed with variable nucleotide sequences, which makes it possible to differentiate one species from another by checking their melting temperature following PCR amplification. The universal primers (panFg) covering entire ITS2 region, from 5.8S to 28S rRNA genes, were designed to differentially identify most Candida species with RTPCR-MTA procedure. Nucleic acids from five genomes of closely related Candida species, which were experimentally spiked into human blood, were extracted and amplified. PCR amplicons were called for melting temperature of Candida albicans (87.49°C), C. glabrata (86.85°C), C. krusei (90.24°C), C. parapsilosis (86.22°C), and C. tropicalis (86.08°C). The melting temperature of each amplicon was consistent and reproducible in three replicate experiments (SD ± 0.04-0.32). The new RTPCR-MTA methodology showed promise in differential diagnosis of closely related Candida species from environmental and clinical samples.

Immunization, hybridoma generation, and selection for monoclonal antibody production.

Monoclonal antibodies (MAbs) produced by a single clone of cells with homogeneous binding specificity for an antigenic determinant have been used in diagnostics and therapeutics. Many new methods have been devised by scientists for making hybridomas and MAbs. The three major steps for producing MAbs are immunization, immortalization, and isolation. Here, we describe technical details of the three important steps for generating mouse hybridomas and MAbs.

Affinity maturation of monoclonal antibodies by multi-site-directed mutagenesis.

High-affinity antibodies are crucial for development of monoclonal antibody (MAb)-based therapeutics for human diseases. Many new detailed methods for affinity maturation have been developed to improve MAb qualities by site-directed mutagenesis, chain shuffling, and error-prone PCR. Site-directed mutagenesis on hotspots in variable heavy (VH) complementary-determining region (CDR) 3 is a commonly used method for improving therapeutic potency and efficacy of targeted MAbs. Strategies for affinity maturation via multi-site-directed mutagenesis in VH-CDR3 described here are for valuable technical tool in the armamentarium of immunologists for development of fast-performance MAbs. Our strategy includes (1) selection of targeted MAb, (2) replacement of certain amino acid residues (e.g., negative or neutral charge to positive amino acids) in VH-CDR3, and (3) determination of binding activity to a target antigen.

Antibacterial efficacy testing of a bioelectric wound dressing against clinical wound pathogens.

Silver-based wound dressings have been developed for the control of bioburden in wounds. However, the popularity and extensive use of silver-based dressings has been associated with emerging microbial resistances to silver. In this study we examined in vitro antibacterial efficacy of a bioelectric dressing containing silver and zinc against various wound pathogens. Antibiotic-sensitive clinical wound isolates showed a 100% reduction in bacterial growth, except that Enterococcus faecalis isolate was shown to survive with a bacterial log10 reduction rate of less than 10(2) CFU. We also investigated antibacterial efficacy against the extended spectrum ß-lactamase (ESBL) bacteria, multidrug-resistant (MDR) bacteria, and methicillin-resistant Staphylococcus aureus (MRSA). The bioelectric dressing was effective in killing wound pathogens including ESBL, MDR, and MRSA in vitro. Furthermore, based on the primary results against E. faecalis, we carried out extensive studies against several nosocomial Enterococcus species including vancomycin-resistant species. Overall, the vancomycin-sensitive or -resistant Enterococcus species were resistant to this dressing at up to 48 h, except for the vancomycin-resistant Enterococcus raffinosus isolate only showing a 100% bacterial reduction at 48 h, but not at 24 h. The results demonstrated the effective bactericidal activity of a bioelectric dressing against antibiotic-sensitive and MDR strains, but Enterococcus species are bacteriostatic.

Pseudomonas aeruginosa induces pigment production and enhances virulence in a white phenotypic variant of Staphylococcus aureus.

Staphyloxanthin is a virulence factor which protects Staphylococcus aureus in stress conditions. We isolated two pigment variants of S. aureus and one strain of Pseudomonas aeruginosa from a single wound infection. S. aureus variants displayed white and yellow colony phenotypes. The sequence of the operons for staphyloxanthin synthesis indicated that coding and promoter regions were identical between the two pigment variants. Quorum sensing controls pigment synthesis in some bacteria. It is also shown that P. aeruginosa quorum-sensing molecules affect S. aureus transcription. We explored whether the co-infecting P. aeruginosa can affect pigment production in the white S. aureus variant. In co-culture experiments between the white variants and a selected number of Gram-positive and Gram-negative bacteria, only P. aeruginosa induced pigment production in the white variant. Gene expression analysis of the white variant did not indicate upregulation of the crtM and other genes known to be involved in pigment production (sigB, sarA, farnesyl pyrophosphate synthase gene [FPP-synthase], hfq). In contrast, transcription of the catalase gene was significantly upregulated after co-culture. P. aeruginosa-induced pigment synthesis and catalase upregulation correlated with increased resistance to polymyxin B, hydrogen peroxide, and the intracellular environment of macrophages. Our data indicate the presence of silent but functional staphyloxanthin synthesis machinery in a white phenotypic variant of S. aureus which is activated by a co-infecting P. aeruginosa via inter-species communication. Another S. aureus virulence factor, catalase is also induced by this co-infecting bacterium. The resulting phenotypic changes are directly correlated with resistance of the white variant to stressful conditions.

Tumor-infiltrating immune cells promoting tumor invasion and metastasis: existing theories.

It is a commonly held belief that infiltration of immune cells into tumor tissues and direct physical contact between tumor cells and infiltrated immune cells is associated with physical destructions of the tumor cells, reduction of the tumor burden, and improved clinical prognosis. An increasing number of studies, however, have suggested that aberrant infiltration of immune cells into tumor or normal tissues may promote tumor progression, invasion, and metastasis. Neither the primary reason for these contradictory observations, nor the mechanism for the reported diverse impact of tumor-infiltrating immune cells has been elucidated, making it difficult to judge the clinical implications of infiltration of immune cells within tumor tissues. This mini-review presents several existing hypotheses and models that favor the promoting impact of tumor-infiltrating immune cells on tumor invasion and metastasis, and also analyzes their strength and weakness.