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The mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest due to their diverse roles in biological systems and are far from being completely understood. Even though cellular cargo releases mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study, this aspect is investigated in detail using Raman microspectroscopic imaging. Considerable evidence is provided to suggest that the release of antibiotic penicillin by the filamentous fungus Penicillium chrysogenum involves EVs. Further, the study also reveals morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs. The results suggest a possible general role of EVs in the release of antibiotics from the producing organisms. [ABSTRACT FROM AUTHOR]

Purpose: We assessed the effect of rebamipide ophthalmic solution on corneal epithelial injury due to benzalkonium chloride (BAK) by fluorescein (FL) staining and corneal resistance (CR). Methods: After determining the absence of corneal epithelial damage by FL and CR, rebamipide ophthalmic solution (50 μL) was instilled five times, each interspaced by 5 min, into one eye of mature New Zealand white rabbits, and likewise physiological saline was instilled into the contralateral eye as the control. After 30 min, eyes were similarly treated with one of the following solutions: BAK solution 0.02%, latanoprost ophthalmic solution (0.02% BAK), or latanoprost ophthalmic solution without BAK. The presence of corneal epithelial damage was quantitated at 10, 30, and 60 min by CR after the last instillation. FL staining was also performed at 60 min after the last instillation. Results: CR ratios (%) at 60 min after the last instillation in rebamipide/BAK and rebamipide/latanoprost (0.02% BAK) groups were significantly increased by 18.3% and 25.6% compared with saline/BAK and saline/latanoprost (0.02% BAK) groups, respectively (P < 0.05). Findings by FL staining were consistent with those by CR; BAK and latanoprost with BAK groups were positive, and eyes with the most severe area and density of corneal epithelial damage (A2D2) were in the saline/BAK group. Conclusion: The rebamipide ophthalmic solution reduces the severity of corneal epithelial injury caused by BAK, an ophthalmic solution preservative. [ABSTRACT FROM AUTHOR]

Alcaligenes faecalis is the predominant Gram‐negative bacterium inhabiting gut‐associated lymphoid tissues, Peyer's patches. We previously reported that an A. faecalis lipopolysaccharide (LPS) acted as a weak agonist for Toll‐like receptor 4 (TLR4)/myeloid differentiation factor‐2 (MD‐2) receptor as well as a potent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as a safe adjuvant. In this study, we characterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis. We synthesized three lipid A molecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1‐ and 4′‐phosphates. Hexaacylated A. faecalis lipid A showed moderate agonistic activity towards TLR4‐mediated signaling and the ability to elicit a discrete interleukin‐6 release in human cell lines and mice. It was thus found to be the active principle of the LOS/LPS and a promising vaccine adjuvant candidate. [ABSTRACT FROM AUTHOR]

Accumulating evidence has revealed that lymphoid tissue-resident commensal bacteria (e.g. Alcaligenes spp.) survive within dendritic cells. We extended our previous study by investigating microbes that persistently colonize colonic macrophages. 16S rRNA-based metagenome analysis using DNA purified from murine colonic macrophages revealed the presence of Stenotrophomonas maltophilia. The in situ intracellular colonization by S. maltophilia was recapitulated in vitro by using bone marrow-derived macrophages (BMDMs). Co-culture of BMDMs with clinically isolated S. maltophilia led to increased mitochondrial respiration and robust IL-10 production. We further identified a 25-kDa protein encoded by the gene assigned as smlt2713 (recently renamed as SMLT_RS12935) and secreted by S. maltophilia as the factor responsible for enhanced IL-10 production by BMDMs. IL-10 production is critical for maintenance of the symbiotic condition, because intracellular colonization by S. maltophilia was impaired in IL-10-deficient BMDMs, and smlt2713 -deficient S. maltophilia failed to persistently colonize IL-10-competent BMDMs. These findings indicate a novel commensal network between colonic macrophages and S. maltophilia that is mediated by IL-10 and smlt2713. [ABSTRACT FROM AUTHOR]

Our previous study demonstrated an indispensable role of Peyer's patches (PPs) for the induction of antigen-specific secretory (S)IgA antibody responses after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin (rS almonella -Tox C). In this study, we defined the PP lymphoid structures and immune cells required for the induction of mucosal SIgA antibody responses. Adoptive transfer of mononuclear cells (MNCs) from PPs into PP-deficient (PP-null) mice failed to elicit tetanus toxoid (TT)-specific mucosal immunity. However, when the same PP MNCs were transferred into lethally irradiated PP-normal recipient mice, PP MNCs preferentially emigrated to recipient PPs, leading to PP lymphoid structures and TT-specific SIgA antibody responses. Significantly reduced numbers of TT-specific IgA antibody-forming cells were detected in the mesenteric lymph nodes (MLNs) and intestinal lamina propria of mice when surface expression of the sphingosine 1-phosphate receptor on lymphocytes was inhibited by its agonist FTY720. However, FTY720 treatment did not alter dendritic cell migration or Salmonella dissemination into these tissues. When r Salmonella -Tox C-stimulated CD4 + T cells isolated from PPs, MLNs and the spleen were co-cultured with B cells from these tissues, significantly increased levels of TT-specific IgA antibody responses were exclusively induced in cultures containing PP B cells. Furthermore, surface IgA + PP B cells produced TT-specific IgA antibody responses in vitro. These findings suggest that PP lymphoid structures and surface IgA + PP B cells are essential elements for the induction of antigen-specific intestinal SIgA antibody responses to oral Salmonella. [ABSTRACT FROM AUTHOR]

Periocular povidone-iodine (PI) and polyvinyl alcohol-iodine (PAI) have had a major role in the prevention of endophthalmitis. The purpose of this study was to investigate the corneal epithelial toxicity of PAI in a rabbit eye model using corneal resistance (CR) measurement, which is a good indicator of cell barrier function. Rabbit eyes were administered PAI solution at 4-, 6-, 8-, or 16-fold dilution with physiological saline solution (saline) or saline alone (control), to the conjunctival sac with/without wash-out with saline. Corneal epithelial injury assessed by fluorescein staining and the CR ratio was measured at 10 minutes (min) to 96 hours (h) after the initial administration. Histological observation was performed in the eyes following the PAI or control administrations. At 120 min after administration of PAI solution, the CR ratio was decreased and superficial punctate keratopathy (SPK) was significantly increased in each of the PAI-administered groups compared to the control. Recovery of CR and SPK after administration of 6- or 8-fold dilution of PAI was significantly delayed in eyes that were not subsequently washed with saline compared with eyes that were. Pre- or post-instillation of 2% rebamipide ophthalmic suspension significantly reduced PAI induced-SPK and -decrease of CR ratio. The CR method was able to accurately and quantitatively evaluate fine corneal epithelial injury. It is suggested that eyes should be washed with saline solution after administration of PAI solution or the instillation of rebamipide to prevent or reduce corneal epithelial injury. [ABSTRACT FROM AUTHOR]

Purpose. This study investigated the effects of oral propolis on the progression of galactose-induced sugar cataracts in rats and the in vitro effects of propolis on high-glucose-induced reactive oxygen species (ROS) and cell death in cultured rat lens cells (RLECs). Methods. Galactose-fed rats and RLECs cultured in high glucose (55 mM) medium were treated with propolis or vehicle control. Relative lens opacity was assessed by densitometry and changes in lens morphology by histochemical analysis. Intracellular ROS levels and cell viability were measured. Results. Oral administration of propolis significantly inhibited the onset and progression of cataract in 15% and 25% of galactose-fed rats, respectively. RLECs cultured with high glucose showed a significant increase in ROS expression with reduced cell viability. Treatment of these RLECs with 5 and 50 μg/mL propolis cultured significantly reduced ROS levels and increased cell viability, indicating that the antioxidant activity of propolis protected cells against ROS-induced damage. Conclusion. Propolis significantly inhibited the onset and progression of sugar cataract in rats and mitigated high-glucose-induced ROS production and cell death. These effects may be associated with the ability of propolis to inhibit hyperglycemia-evoked oxidative or osmotic stress-induced cellular insults. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the safety of five types of antiglaucoma prostaglandin analog ophthalmic formulations, and to clarify their differences in accordance with contained additives (preservatives and surface-active agents). Methods: The following five types of ophthalmic solutions and three types of additives were investigated: latanoprost (Xalatan®; latanoprost), taluprost (Tapros®; taluprost), bimatoprost (Lumigan®; bimatoprost), travoprost (Travatan®; travoprost), travoprost (Travatan Z®; travoprost-Z), benzalkonium chloride (BAK), polyoxyethylene hardening castor oil 40 (HCO-40), and polysorbate 80 (P-80). These experimental solutions were exposed to the cultured cells of a rabbit-derived corneal cell line for a certain time, and the exposure time causing 50% cell damage (CD50), indicated by the ratio of viable cells to total cells was calculated (in vitro). In addition, corneal resistance (CR) was measured and CR ratio (post-treatment CR/pretreatment CR x 100) was calculated (in vivo). Results: CD50 of each ophthalmic solution was the longest with taluprost, followed by travoprost-Z, bimatoprost, travoprost, and latanoprost. CD50 of 0.005%, 0.01%, and 0.02% BAK was 14.5 minutes, 8.1 minutes, and 4.0 minutes, respectively. The number of viable cells decreased to 60%, 8 minutes after exposure with HCO-40, and 30 minutes after being exposed to P-80. The CR ratio was 81.0% with travoprost and 82.0% with latanoprost, indicating a significant posttreatment reduction of CR (P , 0.05). The CR ratio did not decrease after treatment with taluprost, travoprost-Z, or bimatoprost. The CR ratio of 0.005%, 0.01%, and 0.02% BAK was 105.0%, 90.5%, and 68.7%, respectively, and that of HCO-40 and P-80 was 108.7% and 114.2%, respectively. Conclusion: BAK, HCO-40, and P-80 were thought to be involved in corneal injuries caused by each ophthalmic solution. Corneal injuries due to surface action were observed when using HCO-40 and P-80. When HCO-40 was combined with BAK, it induced micellar BAK and reduced corneal injuries by BAK. [ABSTRACT FROM AUTHOR]

Aim: It was the aim of this study to compare the pharmacokinetics of moxifloxacin (MFLX) hydrochloride in rabbits after topical and oral administration. Methods: Three 50-μl applications of MFLX (0.5%) topical ophthalmic solution were instilled into the cul-de-sac of New Zealand white rabbits at 15-min intervals. Aqueous and vitreous samples were collected and analyzed 30-240 min after the final instillation. Assays were performed using high-performance liquid chromatography. MFLX (16 mg/kg of body weight) was administered orally. Drug concentrations in aqueous, vitreous and serum samples, collected at 30-360 min after administration, were determined using high-performance liquid chromatography. Results: After topical administration, the maximum concentrations of MFLX in the aqueous and vitreous samples were 10.2 ± 1.6 μg/ml (30 min; n = 6) and 0.10 ± 0.03 μg/ml (30 min; n = 6), respectively. After oral administration, the maximum concentrations in the aqueous, vitreous and serum samples were 0.9 ± 0.3 μg/ml (120 min; n = 6), 0.7 ± 0.2 μg/ml (240 min; n = 6) and 1.6 ± 0.9 μg/ml (120 min; n = 6), respectively. The percentages of serum MFLX concentration in the aqueous and vitreous samples after oral administration were 55.2 and 41.7%, respectively. Conclusions: The aqueous concentration of MFLX was about 10-fold higher after topical than after oral administration. However, intravitreal MFLX concentrations after oral administration were about 7-fold higher than those after topical administration. The MFLX concentrations in the aqueous humor following oral administration exceeded the minimum inhibitory concentration for 90% of the bacteria involved in ocular infection. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)β-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFβ-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN (hDCN-Tg). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFβ-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation. [ABSTRACT FROM AUTHOR]

Lymphoid-tissue-resident commensal bacteria (LRCs), including Alcaligenes faecalis , are present in intestinal lymphoid tissue including the Peyer's patches (PPs) of mammals and modulate the host immune system. Although LRCs can colonize within dendritic cells (DCs), the mechanisms through which LRCs persist in DCs and the symbiotic relationships between LRCs and DCs remain to be investigated. Here, we show an intracellular symbiotic system in which the LRC Alcaligenes creates a unique energy shift in DCs. Whereas DCs showed low mitochondrial respiration when they were co-cultured with Escherichia coli , DCs carrying A. faecalis maintained increased mitochondrial respiration. Furthermore, E. coli induced apoptosis of DCs but A. faecalis did not. Regarding an underlying mechanism, A. faecalis -unlike E. coli -did not induce intracellular nitric oxide (NO) production in DCs due to the low activity of its lipopolysaccharide (LPS). Therefore, A. faecalis , an example of LRCs, may persist within intestinal lymphoid tissue because they elicit little NO production in DCs. In addition, the symbiotic DCs exhibit characteristic physiologic changes, including a low rate of apoptosis and increased mitochondrial respiration., (Copyright © 2020 Hosomi, Shibata, Shimoyama, Uto, Nagatake, Tojima, Nishino, Takeyama, Fukase, Kiyono and Kunisawa.)

Mucosal surfaces in the body, especially the intestine, are constantly exposed to trillions of microbiomes. Accumulating evidence has revealed that changes in the composition of the gut microbiome, especially that of the commensal bacteria population, are frequently associated with immunologic disorders. These changes coincide with changes in the production of certain dietary metabolites. Recent studies have uncovered the molecular and cellular mechanisms underlying the relationships among diet, commensal bacteria, and the host immune system. In this review, we describe how dietary and microbial metabolites modulate host immunity.

Interleukin-22 (IL-22) acts protectively and harmfully on intestinal tissue depending on the situation; therefore, IL-22 signaling needs to be tightly regulated. IL-22 binding protein (IL-22BP) binds IL-22 to inhibit IL-22 signaling. It is expressed in intestinal and lymphoid tissues, although its precise distribution and roles have remained unclear. In this study, we show that IL-22BP is highly expressed by CD11b + CD8α - dendritic cells in the subepithelial dome region of Peyer's patches (PPs). We found that IL-22BP blocks IL-22 signaling in the follicle-associated epithelium (FAE) covering PPs, indicating that IL-22BP plays a role in regulating the characteristics of the FAE. As expected, FAE of IL-22BP-deficient ( Il22ra2 - /- ) mice exhibited altered properties such as the enhanced expression of mucus and antimicrobial proteins as well as prominent fucosylation, which are normally suppressed in FAE. Additionally, Il22ra2 - /- mice exhibited the decreased uptake of bacterial antigens into PPs without affecting M cell function. Our present study thus demonstrates that IL-22BP promotes bacterial uptake into PPs by influencing FAE gene expression and function., (© 2017 Jinnohara et al.)

Background: Thymic lymphoid hyperplasia is often present with myasthenia gravis as well as other autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Of the 4 cases of thymic lymphoid hyperplasia associated with Sjögren syndrome that have been reported, no case with a thymic lesion diagnosis that led to the diagnosis of Sjögren syndrome has been reported. We herein report a case of thymic lymphoid hyperplasia with multilocular thymic cysts, diagnosed before Sjögren syndrome., Case Presentation: A 37-year-old Japanese woman had an approximate 5-cm anterior mediastinal mass detected by chest imaging. The resected lesion revealed multilocular thymic cysts that were filled with colloid-like material. Histology showed lymph follicular hyperplasia with many epithelial cysts. The epithelium consisted of thymic medullary epithelium, and no epithelial proliferation was seen in the lymphoid tissue. Lymphocytes were composed of an organized mixed population of mature T and B cells without significant atypia. The infiltrated B cells did not reveal light chain restriction or immunoglobulin heavy chain gene rearrangement. After the pathological diagnosis of thymic lesion, tests for the presence of autoantibodies were positive for antinuclear antibodies, rheumatic factor, and anti-SSA/Ro antibodies. The Schirmer's, chewing gum, and Saxon tests showed decreased salivary and lacrimal secretion. Lip biopsy showed focal lymphocytic sialadenitis. The signs and symptoms of Sjögren syndrome had not resolved, without aggravation, 1 year after the thymectomy., Conclusion: When a case with thymic lymphoid hyperplasia without myasthenia gravis is encountered, it is essential to consider the presence of another autoimmune disease including Sjögren syndrome.

Background: Peyer's patches (PPs) play a major role in mucosal immunity. However, their roles in nonsteroidal anti-inflammatory drug-induced enteropathy are poorly understood., Methods: Wild-type (WT) and PP-null mice were injected with indomethacin. Twenty-four hours later, the cellular profiles and cytokine levels in the PPs, mesenteric lymph nodes (MLNs), and lamina propria (LP) of the small intestine were measured. WT and PP-null mice were given antibiotics before indomethacin treatment to evaluate enteropathy. Naive CD4 T cells were co-cultured with CD103 or CD103 dendritic cells (DCs) to analyze the interleukin (IL)-10 expression levels. Finally, WT mice adoptively transferred with CD103 or CD103 DCs were injected with indomethacin., Results: The proportion of CD103 DCs in PPs and MLNs and IL-10-expressing CD4 T cells of PPs and the LP increased after indomethacin treatment. The PP-null mice showed greater indomethacin-induced enteropathy, fewer CD103 DCs in their MLNs, and lower proportion of IL-10-expressing CD4 T cells of their LP than WT mice, regardless of commensal bacteria. Naive splenic CD4 T cells co-cultured with CD103 DCs isolated from the MLNs of indomethacin-injected WT mice produced a higher amount of IL-10 compared with those co-cultured with CD103 DCs. Moreover, WT mice that received CD103 DCs showed milder enteropathy than those that received CD103 DCs., Conclusions: PPs play a protective role in nonsteroidal anti-inflammatory drug-induced enteropathy, and this protection is associated with an increase in CD103 DCs and IL-10-producing CD4 T cells in the intestine, independent of the commensal bacteria.

Peyer's patches (PPs) simultaneously initiate active and quiescent immune responses in the gut. The immunological function is achieved by the rigid regulation of cell distribution and trafficking, but how the cell distribution is maintained remains to be elucidated. In this study, we show that binding of stromal cell-derived lymphoid chemokines to conventional dendritic cells (cDCs) is essential for the retention of naive CD4(+) T cells in the interfollicular region (IFR) of PPs. Transitory depletion of CD11c(high) cDCs in mice rapidly impaired the IFR structure in the PPs without affecting B cell follicles or germinal centers, lymphoid chemokine production from stromal cells, or the immigration of naive T cells into the IFRs of PPs. The cDC-orchestrated retention of naive T cells was mediated by heparinase-sensitive molecules that were expressed on cDCs and bound the lymphoid chemokine CCL21 produced from stromal cells. These data collectively reveal that interactions among cDCs, stromal cells, and naive T cells are necessary for the formation of IFRs in the PPs.

Both SP-D and KL-6/MUC1 are established biomarkers of the interstitial pneumonias, including idiopathic pulmonary fibrosis (IPF), but the causes and clinical outcomes based on their independent effects are not known. Eleven asymptomatic patients, detected with honeycombing on high-resolution computed tomography (HRCT), were compared with 17 other IPF outpatients having slight respiratory symptoms and honeycombing as well. Although SP-D was increased in both groups, KL-6 was significantly higher in the symptomatic IPF group. When the patients (n = 11) having both biomarkers elevated were compared with the other patients (n = 6) with only SP-D elevated, the distribution of fibrotic lesions with honeycombing on HRCT was larger and the survival time was shorter in the patients having both biomarkers elevated. Immunohistochemical analysis also differentiated these biomarkers in the lung. These results suggest both a cause and the prognostic value of dissociation of these biomarkers.

The pathogenesis of acute exacerbation of idiopathic pulmonary fibrosis (IPF) remains to be elucidated. To evaluate the roles of inflammatory mediators in acute exacerbation, the concentrations of high mobility group protein B1 (HMGB1), a chief mediator of acute lung injury, and 18 inflammatory cytokines were measured in the bronchoalveolar lavage fluid, serially sampled from seven IPF patients after the onset of acute exacerbation. HMGB1 gradually increased in the alveolar fluid after the onset of acute exacerbation, in positive correlation with monocytes chemotactic protein-1 (MCP-1), a potent fibrogenic mediator. In the lung tissues of eight IPF patients autopsied after acute exacerbation, intense cytoplasmic staining for HMGB1 was observed in the alveolar epithelial cells in alveolar capillary augmented lesions, where the capillary endothelial cells remarkably reduced the expression of thrombomodulin, an intrinsic antagonist of HMGB1. These results suggest pathogenic roles for HMGB1 and MCP-1 in the late phase of acute exacerbation of IPF.

Background: The lymphatics in the interlobular and subpleural parenchyma contribute to alveolar clearance in the lung, but the information on the remodeling of these lymphatics is quite limited in idiopathic pulmonary fibrosis lungs that contain severe fibrosis in these regions. We compared the alteration of these lymphatics and lymphangiogenesis among idiopathic pulmonary fibrosis and nonfibrotic interstitial pneumonias with a better prognosis., Methods and Results: The lung tissue specimens of eighteen patients with idiopathic pulmonary fibrosis (ten surgical biopsies and eight autopsies), six with organizing pneumonia, six with cellular nonspecific interstitial pneumonia, and five normal controls were examined by morphometric analysis of the lymphatics identified by immunohistochemistry. In addition, three-dimensional reconstruction of lymphatics, apoptosis of lymphatic endothelial cells and the cells producing growth factors for lymphangiogenesis were also evaluated. Both the subpleural and the interlobular lymphatics in idiopathic pulmonary fibrosis lungs were significantly decreased in the severe fibroconnective lesions, with rare lymphangiogenesis. The three-dimensional images of the subpleural lymphatics in idiopathic pulmonary fibrosis clearly revealed destruction by fibrosis; apoptosis was observed in these lymphatic endothelial cells. In contrast, organizing pneumonia and cellular nonspecific interstitial pneumonia preserved these lymphatics, and active lymphangiogenesis occurred in the alveolar lesions., Conclusions: These results reveal severe damage of the subpleural and interlobular lymphatics in idiopathic pulmonary fibrosis lungs, and suggest impaired alveolar clearance as another pathogenesis of its refractoriness.

Antibacterial proteins are important participants in the innate immunity system. Elafin and SLPI are the whey acidic protein (WAP) motif proteins with both antibacterial activity and antiprotease activity, and their role in innate immunity is under intense investigation. We cloned two novel antibacterial WAP motif proteins from mice, SWAM1 and SWAM2. SWAM1 and SWAM2 are composed of a signal sequence and a single WAP motif that has high homologies with the WAP motifs of elafin and SLPI. SWAM1 is constitutively expressed in kidney and epididymis, and is induced in the pneumonic lung. SWAM2 is constitutively expressed in tongue. SWAM1 and SWAM2 inhibit the growth of both Escherichia coli and Staphylococcus aureus at a IC(90) (concentration that achieves 90% inhibition) of 10 microM. Human genes LOC149709 and huWAP2 are considered to be human SWAM1 and SWAM2, respectively. These and several WAP motif proteins (WAP1, elafin, SLPI, HE4, eppin, C20orf170, LOC164237, and WFDC3) form a gene cluster on human chromosome 20, suggesting that they may be derived from the same ancestral gene by gene duplication. Our results underscore the role of the WAP motif as a skeletal motif to form antibacterial proteins, and warrant the study of antibacterial activity in other WAP motif proteins.