Identification and Functional Characterization of Peptides With Antimicrobial Activity From the Syphilis Spirochete, Treponema pallidum.

The etiological agent of syphilis, Treponema pallidum ssp. pallidum, is a highly invasive "stealth" pathogen that can evade the host immune response and persist within the host for decades. This obligate human pathogen is adept at establishing infection and surviving at sites within the host that have a multitude of competing microbes, sometimes including pathogens. One survival strategy employed by bacteria found at polymicrobial sites is elimination of competing microorganisms by production of antimicrobial peptides (AMPs). Antimicrobial peptides are low molecular weight proteins (miniproteins) that function directly via inhibition and killing of microbes and/or indirectly via modulation of the host immune response, which can facilitate immune evasion. In the current study, we used bioinformatics to show that approximately 7% of the T. pallidum proteome is comprised of miniproteins of 150 amino acids or less with unknown functions. To investigate the possibility that AMP production is an unrecognized defense strategy used by T. pallidum during infection, we developed a bioinformatics pipeline to analyze the complement of T. pallidum miniproteins of unknown function for the identification of potential AMPs. This analysis identified 45 T. pallidum AMP candidates; of these, Tp0451a and Tp0749 were subjected to further bioinformatic analyses to identify AMP critical core regions (AMPCCRs). Four potential AMPCCRs from the two predicted AMPs were identified and peptides corresponding to these AMPCCRs were experimentally confirmed to exhibit bacteriostatic and bactericidal activity against a panel of biologically relevant Gram-positive and Gram-negative bacteria. Immunomodulation assays performed under inflammatory conditions demonstrated that one of the AMPCCRs was also capable of differentially regulating expression of two pro-inflammatory chemokines [monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8)]. These findings demonstrate proof-of-concept for our developed AMP identification pipeline and are consistent with the novel concept that T. pallidum expresses AMPs to defend against competing microbes and modulate the host immune response.

The clinical potential of circulating immune cell counts in primary gastric lymphoma.

BACKGROUND: High neutrophil-lymphocyte ratio (NLR) is linked to poor overall survival (OS) in gastrointestinal tract cancers. This study explores the clinical value of NLR, in addition to absolute lymphocyte count (ALC) and other hematologic parameters in association with distant metastases and OS in primary gastric lymphoma (PGL) patients. METHODS: Clinical data of 139 PGL patients who received treatment at King Hussein Cancer Center (KHCC), Amman-Jordan were retrospectively evaluated. Using data from complete blood count (CBC) tests, the following hematologic parameters: absolute neutrophil count (ANC), ALC, absolute eosinophil count (AEC), absolute monocyte count (AMC), NLR, platelet-lymphocyte ratio (PLR), and monocyte-lymphocyte ratio (MLR) were assessed in association with the following clinical outcomes: presence or absence of baseline distant metastases and OS. We conducted univariate and multivariate analyses assessing the various hematologic parameters in association with distant metastases. RESULTS: Univariate and multivariate analyses indicated that patients with an elevated NLR (>3.14) displayed more baseline distant metastases compared to patients with a low NLR (≤3.14), (P value: 0.02 and 0.018, respectively). High baseline ALC (>1,819/µL) was associated with lower baseline distant metastases (P value: 0.04). In the OS analysis, high baseline ANC (>5,100/µL), NLR (>2.75), and PLR (>0.16) were associated with poor OS, (P value: 0.027, 0.016, and 0.011 respectively). CONCLUSIONS: High NLR and ALC were associated with baseline distant metastases. High baseline ANC, NLR, and PLR were associated with poor OS. Hematologic parameters might be potentially helpful in assessing and correlating NLR with the response success to treatment in PGL.

The Emerging Role of Epigenetic Mechanisms in the Causation of Aberrant MMP Activity during Human Pathologies and the Use of Medicinal Drugs.

Matrix metalloproteinases (MMPs) cleave extracellular matrix proteins, growth factors, cytokines, and receptors to influence organ development, architecture, function, and the systemic and cell-specific responses to diseases and pharmacological drugs. Conversely, many diseases (such as atherosclerosis, arthritis, bacterial infections (tuberculosis), viral infections (COVID-19), and cancer), cholesterol-lowering drugs (such as statins), and tetracycline-class antibiotics (such as doxycycline) alter MMP activity through transcriptional, translational, and post-translational mechanisms. In this review, we summarize evidence that the aforementioned diseases and drugs exert significant epigenetic pressure on genes encoding MMPs, tissue inhibitors of MMPs, and factors that transcriptionally regulate the expression of MMPs. Our understanding of human pathologies associated with alterations in the proteolytic activity of MMPs must consider that these pathologies and their medicinal treatments may impose epigenetic pressure on the expression of MMP genes. Whether the epigenetic mechanisms affecting the activity of MMPs can be therapeutically targeted warrants further research.

Comparative Serum Analyses Identify Cytokines and Hormones Commonly Dysregulated as Well as Implicated in Promoting Osteolysis in MMP-2-Deficient Mice and Children.

Deficiency of matrix metalloproteinase 2 (MMP-2) causes a complex syndrome characterized by multicentric osteolysis, nodulosis, and arthropathy (MONA) as well as cardiac valve defects, dwarfism and hirsutism. MMP-2 deficient (Mmp2 -/-) mice are a model for this rare multisystem pediatric syndrome but their phenotype remains incompletely characterized. Here, we extend the phenotypic characterization of MMP-2 deficiency by comparing the levels of cytokines and chemokines, soluble cytokine receptors, angiogenesis factors, bone development factors, apolipoproteins and hormones in mice and humans. Initial screening was performed on an 8-year-old male presenting a previously unreported deletion mutation c1294delC (Arg432fs) in the MMP2 gene and diagnosed with MONA. Of eighty-one serum biomolecules analyzed, eleven were upregulated (>4-fold), two were downregulated (>4-fold) and sixty-eight remained unchanged, compared to unaffected controls. Specifically, Eotaxin, GM-CSF, M-CSF, GRO-α, MDC, IL-1ß, IL-7, IL-12p40, MIP-1α, MIP-1ß, and MIG were upregulated and epidermal growth factor (EGF) and ACTH were downregulated in this patient. Subsequent analysis of five additional MMP-2 deficient patients confirmed the upregulation in Eotaxin, IL-7, IL-12p40, and MIP-1α, and the downregulation in EGF. To establish whether these alterations are bona fide phenotypic traits of MMP-2 deficiency, we further studied Mmp2 -/- mice. Among 32 cytokines measured in plasma of Mmp2 -/- mice, the cytokines Eotaxin, IL-1ß, MIP-1α, and MIG were commonly upregulated in mice as well as patients with MMP-2 deficiency. Moreover, bioactive cortisol (a factor that exacerbates osteoporosis) was also elevated in MMP-2 deficient mice and patients. Among the factors we have identified to be dysregulated in MMP-2 deficiency many are osteoclastogenic and could potentially contribute to bone disorder in MONA. These new molecular phenotypic traits merit being targeted in future research aimed at understanding the pathological mechanisms elicited by MMP-2 deficiency in children.

The clinical value of peripheral immune cell counts in pancreatic cancer.

BACKGROUND: Elevated neutrophil-lymphocyte ratio (NLR) is linked to poor overall survival (OS) in pancreatic cancer. We aim to investigate the association of the various hematologic markers, in particular NLR among others, with distant metastases, a common feature in pancreatic cancer. METHODS: Clinical data from 355 pancreatic cancer patients managed at King Hussein Cancer Center (Amman-Jordan) have been reviewed. We examined the relationship between absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute eosinophilic count (AEC), absolute monocytic count (AMC), NLR, monocyte to lymphocyte ratio (MLR) and platelet to lymphocyte ratio (PLR) with the presence of baseline distant metastases and OS. Receiver Operating Characteristic (ROC) curve analysis was plotted to identify the NLR optimum cutoff value indicative of its association with distant metastases. RESULTS: On univariate and multivariate analyses patients whom on presentation had high NLR (≥3.3) showed more baseline distant metastases compared to patients with low NLR (<3.3), (p-value: <0.0001 and <0.0001, respectively). Patients with high baseline ANC (≥5500/µL), AMC (≥600/µL), MLR (≥0.3) had more baseline distant metastases in comparison to patients with lower values (p-value: 0.02, 0.001, and <0.0001, respectively). High ANC, NLR, MLR, and PLR and low ALC were associated with poorer OS, (p-value: <0.0001, <0.0001, <0.0001, 0.04, and 0.01, respectively). CONCLUSION: This study presents additional evidence of the association of some of the hematologic markers; in particular ANC, NLR, AMC, and MLR, with baseline distant metastases and poor outcome in pancreatic cancer. Whether these immune phenomena can help in identifying patients at higher risk for the subsequent development of distant metastases is unknown.

Hematologic markers of distant metastases in gastric cancer.

BACKGROUND: High neutrophil-lymphocyte ratio (NLR) is associated with poor overall survival (OS) in gastric cancer. This study evaluates whether NLR, in addition to other parameters including absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute eosinophil count (AEC), absolute monocyte count (AMC), monocyte-lymphocyte ratio (MLR), and platelet-lymphocyte ratio (PLR) are associated with distant metastases, a common and poor prognostic feature of gastric cancer. METHODS: Clinical data from 502 gastric cancer patients treated at King Hussein Cancer Center (Amman, Jordan) have been retrospectively reviewed. We examined the association between ANC, ALC, AEC, AMC, NLR, MLR and PLR with the baseline distant metastases and OS. Receiver operating characteristic (ROC) curve analysis was utilized to determine the optimal NLR cutoff value for association with distant metastases. RESULTS: Univariate and multivariate analyses showed that patients with high baseline NLR (≥3.9) had more distant metastases on presentation than patients with low NLR (<3.9), (P value: 0.0001 and 0.0005, respectively). Furthermore, patients with high baseline ANC (≥6,015/µL), AEC (≥215/µL), PLR (≥0.15) had more distant metastases in comparison to patients with low baseline ANC (<6,015/µL), AEC (<215/µL), PLR (<0.15) (P value: 0.024, 0.001, and 0.001, respectively). High ANC, NLR, MLR and PLR are associated with poor OS (P value: 0.046, 0.0003, 0.027, and <0.0001, respectively). CONCLUSIONS: High ANC, AEC, NLR, and PLR are associated with distant metastases on presentation in gastric cancer. In the era of cancer immunotherapy, whether these immune phenomena predict the response of gastric cancer to immunotherapy is unknown.

Identification of fibrinogen as a natural inhibitor of MMP-2.

Non-genetic MMP-2 insufficiency is a relatively unexplored condition which could be induced by pathological overexpression of endogenous MMP-2 inhibitors such as TIMPs and/or the acute phase reactant alpha-2-macroglobulin. Here, we investigate the hypothesis that human fibrinogen (FBG) - an acute phase reactant - inhibits human MMP-2. Following an unexpected observation where sera from human donors including arthritis patients with increased levels of serum FBG exhibited reduced binding of serum proMMP-2 to gelatin, we found that human FBG (0 to 3.6 mg/mL i.e., 0 to 10.6 µM) concentration-dependently inhibited human proMMP-2 and MMP2 from binding to gelatin. Moreover, at normal physiological concentrations, FBG (5.29-11.8 µM) concentration-dependently inhibited (40-70% inhibition) the cleavage of fluorescein-conjugated gelatin by MMP-2, but not MMP-9. Indicative of a mixed-type (combination of competitive and non-competitive) inhibition mechanism, FBG reduced the Vmax (24.9 ± 0.7 min-1 to 17.7 ± 0.9 min-1, P < 0.05) and increased the Michaelis-Menten constant KM (204 ± 6 nM to 478 ± 50 nM, P < 0.05) for the reaction of MMP-2 cleavage of fluorescein-conjugated gelatin. In silico analyses and studies of FBG neutralization with anti-FBG antibodies implicated the domains D and E of FBG in the inhibition of MMP-2. In conclusion, FBG is a natural selective MMP-2 inhibitor, whose pathological elevation could lead to MMP-2 insufficiency in humans.