4). In support of this, we found that the treatment of BCG-vaccinated mice with COX2 inhibitor in vivo significantly reduced Ag85B-specific Th17-cell responses (Fig.

4G), but not Ag85B-specific Th1-cell responses (Fig. 4H) or vaccine-induced protection in the lung following M. tuberculosis challenge (Fig. 4I). These data suggest that both in vivo and in vitro, blocking PGE2 results in reduced Th17-cell responses. Importantly, despite reduced Th17-cell responses, a competent Th1-cell response is generated, likely due to coincident loss of IL-10 production that can confer Cilomilast clinical trial vaccine-induced protection. These data suggest a role for IL-17 specifically to overcome IL-10 inhibitory effects. Consistent with a role for IL-17 in the induction of IL-12 in DCs 12, 13, we found that IL-17 treatment of BCG-exposed DCs enhanced IL-12 (Fig. 5A). Importantly, the treatment of IL-17 significantly decreased BCG-induced IL-10 production in DCs

(Fig. 5B). These data suggest that BCG exposure results in the induction of PGE2 and high levels of IL-10 that initially inhibits IL-12 production and Th1-cell Stem Cell Compound Library solubility dmso responses in vivo (Fig. 2). Accordingly, the peak of PGE2 induction in vivo following BCG vaccination was at day 4, with significantly lower levels of PGE2 at later time points (Fig. 5C). However, BCG-induced PGE2 also mediates IL-23 induction and drives subsequent Th17-cell responses. IL-17 then induces IL-12 production and inhibits IL-10 production and mediates IFN-γ responses at later time points. Therefore, IFN-γ protein expression was not detected early, but

at later time points following BCG vaccination in vivo (Fig. 5D). In order to confirm that PGE-dependent IL-17 mediates Th1-cell responses to overcome BCG-mediated IL-10 inhibition, we depleted IL-17 in il10−/− BCG-vaccinated mice and measured Ag85B-specific Th1-cell responses in DLNs. Our data show that the depletion of IL-17 in B6 mice resulted in decreased Ag85B-specific Th1-cell response (Fig. 5E). However, depletion of IL-17 in il10−/− mice BCKDHA did not result in decreased Ag85B-specific Th1-cell responses when compared with il-10−/− BCG-vaccinated mice treated with isotype control antibody (Fig. 5E). These data suggest that IL-17 responses are required to drive Th1-cell responses, specifically to overcome IL-10-dependent Th1-cell inhibitory effects. PGE2 is critical for the induction of IL-23 responses and Th17-cell responses 18, 19, while inhibiting IL-12 responses through the production of IL-10 16. However, since PGE2-matured DCs can effectively induce IFN-γ-producing T cells 29, 30, we show that the immune system has developed means of counteracting the PGE2-mediated suppression of Th1-cell immunity. In this article, we show that the role for mycobacteria-induced PGE 2 is bifunctional since it not only induces IL-10 and limits early Th1-cell response, but also simultaneously induces IL-23 and subsequent IL-17 responses.

Obtained cell clusters were isolated with a 40-μm mesh filter (Becton Dickinson) and magnetically separated into a CD4+ or into CD4+CD25high/– fractions using a Miltenyi MACS® kit according to the suppliers manual. A proportion of the CD25high T-cell population was checked for Foxp3 expression with the purity≥85% in all experiments. Peripheral blood was drawn directly from the heart of sacrificed mice. For CNS-derived lymphocyte flow cytometry, a Percoll density gradient was used as described previously 29. In brief, mice were sacrificed with CO2 and immediately perfused with 10 mL of PBS before harvesting this website the brain and spinal cord. The tissue was,

similar to the lymph nodes, mechanically homogenized in PBS, layered on a 30%/50% Percoll gradient and centrifuged without brake at 600×g for 30 min. After removing the top layer of myelin, lymphocytes were harvested at the Percoll interphase. MBMEC were isolated according to Weidenfeller et al.30. The obtained capillary fragments were seeded onto CollagenIV/fibronectin-coated Selleckchem Roxadustat membranes of transwell inserts (6.5 mm Transwell® Pore Polyester Membrane Insert, pore size 3.0 μm, Corning, 2 inserts/mouse brain). Cells were incubated in DMEM high glucose with 2 mM L-glutamine, 100 U/mL

penicillin, 100 μg/mL streptomycin (PAA), 20% plasma derived bovine serum (First Link), 10 ng/mL basic fibroblast growth factor (Peprotech), 100 ng/mL heparin and 4 μg/mL pyromycin (Sigma-Aldrich) for 3 days followed by an additional 2 days of incubation without pyromycin. At this time, the monolayer reached confluence, which was randomly monitored by TEER measurements

(confluence at TEER plateau). Freshly isolated and magnetically separated fractions of CD4+, CD4+CD25high or CD4+CD25− T cells (6×105/insert) were applied on 3.0-μm pore polyester membrane transwell inserts (Corning) with or without a MBMEC layer grown onto the microporous membrane in RPMI1640 with 100 U/mL penicillin, 100 μg/mL streptomycin (PAA) and 2% B-27 serum free supplement (Gibco). T cells from three compartments were harvested after an incubation period of 18 h. Each transwell insert was removed from the well plate; cells from the upper chamber were collected by transfer of the cell suspension into a new conical and ZD1839 rinsing with PBS two times to ensure removal of all remaining T cells. T cells from within the MBMEC layer were harvested by incubating the cell layer with Accutase (PAA) for 10 min at 37°C and 4% CO2. The cells were then detached by rinsing with PBS and transferred into a new conical. Cells in the lower chamber were collected and wells were subsequently rinsed with PBS twice to ensure complete removal of cells. For quantification, Calibrite beads (Becton Dickinson) were added prior to harvesting the cells. Cell number was determined by counting 1×104 reference beads with a four-color FACSCalibur flow cytometer (Becton Dickinson).

Notably, all of the control (PBS-inoculated) chickens died within 7 days post-challenge, whereas none of the AESN1331-inoculated chickens died. The clinical and lesion scores (in heart and liver) of the AESN1331 group were significantly lower than those of the control group. The challenge strain was detected in the hearts and Opaganib molecular weight livers of all of the dead control chickens, but

only in a subset (20%–50%) of AESN1331-inoculated birds. We constructed and characterized an APEC O78 strain carrying a deletion of the crp gene. The mutant bacterium, AESN1331, had the following favorable characteristics: (i) low pathogenicity for chickens and embryonated eggs; (ii) protection against colibacillosis caused by the avian E. coli O78 wild-type strain, and (iii) ease of inoculation by various routes. Thus, AESN1331 is a suitable candidate for a live vaccine against avian colibacillosis. Our previous study also demonstrated that hemolytic APEC strains produce β-hemolysin, which is encoded

by a gene whose expression is dependent on crp gene function (36). In the present study, we showed that the LD50 value was higher for AESN1331 than for the parent strain and that the in vivo survival time of AESN1331 was clearly shortened. The mutant in this study was phenotypically different from the parent in other respects, including loss of some abilities such as hemolytic activity, adsorption of Congo red, tryptophan deaminase activity, indole production, and utilization of multiple sugars. check As with virulence factor production, these abilities are presumably directly DNA Synthesis inhibitor or indirectly governed by the crp gene product. Peighambari et al. showed that a ΔcyaΔcrp mutant of an APEC O78 strain was not immunogenic against airsacculitis induced by homologous experimental challenge (23, 24). In contrast, a ΔcyaΔcrp mutant of Salmonella Typhimurium provided laying hens with marked protection against colonization and invasion by S. Typhimurium and S. Enteritidis

(42). Our crp deletion mutant of APEC O78 was an effective live vaccine against septicemia caused by experimental challenge with a APEC O78 strain. There appears to be a fine line between reduced virulence and immunogenicity; the behavior of a mutant presumably varies with the species, serovar, strain, and infection model. We postulate that cya crp double mutants of APEC are too attenuated for growth to serve as live vaccines. It seems that direct testing is the only way to assess the immunization potential of various attenuated mutants. Gene reversions are extremely rare in large fragment (351 bp nucleotide) deletion, though calculation of probability indicates the possibility of reversion of our single mutant is higher than that of double mutant. The survival time of our mutant in the field was too short for acquisition of a functional crp gene from exogenous source to be possible.

Many more studies have been done on the human T-cell responses to viral infections in mice with reconstituted human immune system components, particularly on CD8+ T-cell responses. In both HIV and EBV infection of reconstituted mice viral antigen-specific T-cell responses were detected, but their frequency as assessed by IFN-γ production usually did not exceed 0.1%, despite the fact that a substantial proportion of the expanded CD8+ T-cell population could be detected by MHC class I/viral peptide tetramer staining [5, 38, selleck chemical 40, 64, 68]. This inability of most expanded antiviral CD8+ T cells to secrete cytokines might result from infection-induced

differentiation of these cells and concomitant upregulation of the inhibitory receptor PD-1. Indeed, PD-1 blockade was able to rescue proinflammatory cytokine secretion in HIV-infected reconstituted mice [69]. However, this terminal differentiation of the expanded CD8+ T cells might not negatively affect their cytotoxicity, and indeed significant perforin and granzyme B upregulation as well as cytolytic activity was found in expanded CD8+ T cells after HIV and EBV infection [38, 64, 68, 70]. Nevertheless, the viral peptide

epitopes that were recognized by these responding T cells seemed to strongly depend on the MHC class I context, in which the CD8+ T-cell repertoire VX809 is educated in the thymus. In mice with human thymic transplants, the reconstituted CD8+ T-cell compartments can readily recognize immunodominant dengue virus and HIV triclocarban derived epitopes [49, 64]. In reconstituted mice, in which the T-cell repertoire gets selected through the mouse thymus, these immunodominant epitopes are only recognized if the murine host transgenically expresses the respective HLA class I molecules [38, 50, 70]. In the absence of these HLA class I molecules from the murine thymic stroma, presumably unusual and in humans subdominant epitopes are recognized

by the expanding CD8+ T cells. However, this has only been documented for one clonal EBV specificity so far [38]. Although the epitope specificities of the expanding CD8+ T-cell response are still being unraveled in reconstituted mice, this adaptive immune response clearly exerts protective immune control. HIV, for example, accumulates escape mutations in response to primed CD8+ T cells [71]. Moreover, the presence of the protective HLA-B57 molecule on the reconstituted human immune system components and on the thymic transplant allowed better HIV-specific immune control and restricted CD8+ T-cell responses similar to those found in human patients [71].

Furthermore, BMDC treated with rHp-CPI before ovalbumin (OVA) antigen pulsing induced a weaker proliferation response and less interferon-γ production of OVA-specific CD4+ T cells compared with BMDC without rHp-CPI pre-treatment. Adoptive transfer of rHp-CPI-treated and OVA-loaded

BMDC to mice induced significantly lower levels of antigen-specific antibody response than the BMDC loaded with antigen alone. These results demonstrated that the CPI from nematode parasites is able to modulate differentiation and activation stages of BMDC. It also interferes with antigen and MHC-II molecule Ceritinib processing and Toll-like receptor signalling pathway, resulting in functionally deficient DC that induce a suboptimum immune response. Nematode parasite infections are common in many parts of the world and cause significant health problems in humans.[1] Infections with this group of pathogens often undergo a chronic and asymptomatic course and induce a T helper type 2-dominated immune response.[2, 3] In addition, nematode infections often induce immunosuppression, which is believed to be an important strategy for the Z-VAD-FMK solubility dmso survival of the parasite in the host.[4, 5] The immunosuppression associated with nematode infection is also demonstrated as the suppression of immune responses to unrelated

antigens and immune protection against concurrent infection with other pathogens.[6, 7] Epidemiological studies showed that helminth infections in human populations are also associated with decreased prevalence of autoimmune disorders and allergic diseases (hygiene hypothesis).[8, 9] Although nematode infections are known to elicit T helper type 2-dominant immune responses, which are required for immune protection against the nematode pathogens,[10] many

studies show that these pathogens also induce a regulatory T-cell response and cytokines that mediate the immunosuppression.[11-13] CYTH4 In mice infected with the murine nematode parasite, Heligmosomoides polygyrus, we identified a subset of dendritic cells (DC) that are selectively expanded following H. polygyrus infection and induce interleukin-10 (IL-10) production by T cells and FoxP3+ CD4+ T-cell response.[14] Previous studies with H. polygyrus and other nematode species also demonstrated that the crude preparation or excretory–secretory (ES) products from the parasites are able to modulate the phenotypes and functions of immune cells.[15-17] It has been reported that the ES products from H. polygyrus can modulate the antigen presentation function of DC and specifically induce an IL-10-producing T-cell response.[15] However, the immunoregulatory molecule(s) produced by H. polygyrus have not been fully characterized. A number of studies in recent years have shown that cysteine proteases inhibitor (CPI; cystatin) is one of the major immune modulators produced by nematode parasites.

A number of surface receptors coupled to ITAM-bearing adaptors have been shown to regulate myeloid cell functions. Among them, CD300e (IREM-2) appeared selectively expressed

by monocytes and mDC and was shown to associate with DAP12 in transfected cells, delivering activating signals 20. In the present study, we provide data supporting that cross-linking of CD300e triggered the intracellular calcium mobilization and ROS secretion in monocytes. Signaling through CD300e activated monocytes and mDC, promoting their survival and leading to the induction of pro-inflammatory cytokine secretion and increased expression of co-stimulatory molecules. Moreover, CD300e-stimulated mDC enhanced the alloreactive response of CbT cells. Altogether, these results

formally support that CD300e functions as an activating Opaganib receptor capable of regulating the inflammatory and immune responses. The expression pattern and function of CD300e partially differed from other activating myeloid receptors associated to ITAM-bearing adaptors. Unlike the DAP12-associated TREM-1 31, 32, CD300e was not upregulated upon monocyte activation via TLR4 (data not shown), thus resembling the FcRγ-associated receptor hOSCAR 27. CD300e ligation induced a rapid intracellular calcium mobilization, as well as the production of ROS, supporting that this receptor may regulate the microbicidal RAD001 supplier activity of monocytes 33. Similarly, and in line with the previous reports on the ability of both hOSCAR and TREM-1 to trigger the respiratory burst in granulocytes 27, 34, we have observed that TREM-1 activates ROS production also in monocytes. Once recruited and activated at inflammatory sites, monocytes upregulate the expression of co-stimulatory molecules (i.e. CD40, CD83, CD80 and CD86) that, together with cytokine secretion, contribute to T-cell activation

and the generation of an optimal adaptive immune response. Herein, we show that CD300e engagement induced an upregulation of CD25, CD83 and CD86, without detectably influencing the expression of CD40 or CD54, in contrast to TREM-1 31 and hOSCAR activation 27. On the contrary, it is ADP ribosylation factor of note that these two receptors appear capable of triggering the secretion of pro-inflammatory cytokines, including TNF-α and IL-8/CXCL8 in monocytes 27, 31 similarly to CD300e. In our experience, some differences in the functional response patterns were noticed when CD300e was compared with TREM-1 and hOSCAR in monocyte activation assays using specific mAb (Brckalo et al., unpublished data). Yet, it is of note that despite the fact that agonistic mAb are valuable tools to functionally characterize cell surface receptors, data should be cautiously interpreted for comparative analysis between different molecules, unless validated with their natural ligands.

0±1.2 mm, while that for the control strain was 18.5±0.4 mm. This indicates that this website the overexpression of Spx has little effect on

the adaptation of S. epidermidis to diamide. To further confirm this, we extracted the RNA from WT and the spx-overexpressing strain, and determined the transcription level of trxB (encoding thioredoxin reductase and associated with thiol homeostasis). The transcription level of trxB was induced remarkably through the overexpression of Spx in B. subtilis, and decreased in both B. subtilis and S. aureus spx mutants. Consistent with our phenotypic observation, no significant difference of the trxB transcriptional level was found between the spx-overexpressing strain and WT. In addition, we found that the overexpression of Spx has little effect on the stress response of S. epidermidis to ethanol or hydrogen peroxide (data not shown). Spx is a conserved protein in low-G+C-content ACP-196 manufacturer gram-positive bacteria. Its cellular level is controlled by ClpP protease. In both B. subtilis and S. aureus, Spx functions as a novel type of transcriptional regulator, and was proved as a substrate of ClpP protease (Nakano et al., 2002, 2003b; Pamp et al., 2006). Many bacterial functions are regulated by

Spx, such as competence, thiol homeostasis and biofilm formation (Zuber, 2004; Pamp et al., 2006). We found a much higher level of Spx in the clpP mutant strain, suggesting that Spx is likely a substrate of ClpP protease in S. epidermidis. The spx-overexpressing plasmid was constructed by modification of the C-terminal of spx gene, which also supports this view. In S. aureus, Spx negatively regulates biofilm formation (Pamp et al., 2006). In our study, decreased biofilm formation was found in the S. epidermidis Spx-overexpressing Palbociclib mouse strain. The primary attachment and the PIA production were severely reduced in the Spx-overexpressing strain compared with WT, and the biofilm by the strain carrying the antisense spx plasmid was decreased compared with WT. The transcription of atlE was similar to WT in the Spx-overexpressing strain, indicating that Spx does not affect the primary attachment through inhibiting the transcription of atlE.

Olson et al. (2006) have previously found that PIA enhanced the adherence of S. epidermidis to several orthopedic prosthetic biomaterials, including zirconia, ultra-high-molecular-weight polyethylene and cobalt chromium, but had no impact on the primary attachment to polymethylmethacrylate and titanium. In our study, there was no notable difference in the level of the initial adherence between WT and the isogenic PIA-negative strain under the selected experimental conditions. Thus, the mechanism behind the decreased initial attachment of the Spx-overexpressing strain needs further investigation. Decreased icaADBC transcription was found in the Spx-overexpressing strain of S. epidermidis, indicating that Spx affects PIA production by regulating the transcription of icaADBC. In S.

For example, Th17-cell priming requirements have elicited disputes, primarily due to inconsistencies between mouse and human cytokine requirements and in particular due to the controversial role of TGF-β in Th17-cell differentiation [65]. Although Th-cell polarization is a multilayered process that is dependent on signal strength and

the engagement of different co-stimulatory molecules following antigen processing, and the establishment of a complex immunological synapse, the focus of interest has been on cytokine requirements. Most of the approaches to dissect Th17 priming conditions have therefore used polyclonal stimulation of naïve Ruxolitinib manufacturer T cells with anti-CD3 and anti-CD28 antibodies in the presence of well-defined cytokine combinations in vitro. However, human Th17-cell polarization following antigen-specific stimulation with microbes has recently revealed that priming requirements differ, depending on microbial antigen selleck specificity even within the same class of Th cells [12]. Microbial ligands that generate Th17-cell responses through TLR and CLR signaling have primarily, although not exclusively, been defined for C. albicans [66, 67]. Fungal components have been shown to bind to

Dectin1, Dectin2, and Mincle expressed on APCs, which leads to the recruitment of the tyrosine kinase Syk, activation of the adaptor CARD9, and finally to secretion of IL-23, IL-1, IL-6 [66, 67], which are involved in the generation of human Th17 cells. Interestingly, the generation of C. albicans-specific human Th17 cells has been shown to be highly dependent on IL-1β, while S. aureus-specific Th17 cells can be primed in its absence [12]. This not only indicates different pathways for the generation of human Th17 cells but also a strong link between microbial antigen specificities of Th cells with their respective priming requirements.

This has important consequences for the functionality of Th17 cells, since C. albicans-specific, and thus IL-1β-dependent Th17 cells have been shown to co-express IL-17 and IFN-γ but not IL-10, while S. aureus-specific Th17 cells have been shown to be IFN-γ negative but IL-10 positive [12]. IL-1β therefore acts as a molecular switch factor for the generation of pro- versus anti-inflammatory Ketotifen Th17-cell properties [3, 68]. A model disease to exemplify the two-sided interactions of environment and Th cells is chronic mucocutaneous candidiasis, a rare disease characterized by chronic and persistent infection of skin and mucosa with Candida species [69]. Numerous mutations affecting the differentiation and function of Th17 cells have been described for chronic mucocutaneous candidiasis. Namely, humans with loss-of-function mutations in CARD9 and STAT3 or gain-of-function mutations in STAT1 have reduced Th17 cells [70-72]. In other families, IL-17 or its receptor is mutated, or autoantibodies against IL-17 are secreted [73, 74].

We speculate that if Vpu can be presented in a manner that elicits functional and effective ADCC responses, then the Vpu ADCC epitopes that we describe Tamoxifen supplier could be interesting vaccine antigens. Interestingly, a study by Chen et al. in 2003[38] suggested that Vpu-specific antibody responses detected by Western blot were associated with slower disease progression. An important caveat of this work is that our mapping of ADCC responses was limited to linear peptide epitopes that could be defined with individual

peptides. Conformational ADCC epitopes within Vpu and other HIV proteins recognized by LTSP subjects would also be of considerable interest, but such epitopes are more difficult to map. Further, the number of LTSP subjects that generated Vpu peptide-specific ADCC responses was modest (seven of the 65 subjects, 10·8%). However, this might be expected because multiple other mechanisms, such as HLA

class I molecules and CCR5 deletions, have been associated with slow HIV progression.[39, 40] Indeed, 35% of the LTSP subjects tested were CCR5Δ32 heterozygotes and 41% of the LTSP subjects tested had either HLA B27 or B57 alleles. It is possible that ADCC responses targeting common epitopes in Env or other HIV-1 proteins are also associated with slowly progressive HIV. The C1 region of Env has recently been shown to be a common target of ADCC antibodies[41] and we recently showed that ADCC epitopes within C1 can force immune escape.[42] Our ability to fully map Env-specific ADCC in the LTSP cohort was limited by the volumes of sera available from the LTSP cohort and the large number of overlapping peptides spanning Env. selleck screening library This is a subject of ongoing research. The large diversity of infecting Env strains, the ability of Env to readily escape antibody responses, and the limited apparent fitness costs of Env variants potentially makes Env a less attractive target than more conserved HIV proteins.[42-45] Although this study identifies an immune response associated with slow

HIV progression, this does not prove that this response is causally linked to slow progression. LTSP subjects are by definition infected for long periods of time and the anti-HIV ADCC responses may broaden over Baricitinib time unrelated to the control of viraemia. Previous smaller studies suggest broadening of ADCC responses over time.[46, 47] However, we are now in a position to definitively test the protective effects of these Vpu ADCC antibodies in passive transfer studies in macaques subsequently challenged with chimeric SHIV expressing HIV-1 Vpu. Previous passive transfer experiments using neutralizing antibodies have suggested an important additive role for ADCC functions,[10, 48] but the utility of ADCC antibodies without neutralizing activity in protecting macaques from SHIV infection is not known. In conclusion, we studied HIV-specific ADCC responses in a large cohort of LTSP subjects.

There were no serious systemic complications. Although we have described limited cases and supporting data are lacking, we find more feel that this procedure might

be useful for microsurgical reconstruction of the lower limb. © 2010 Wiley-Liss, Inc. Microsurgery 30:376–379, 2010. “
“Venous flow-through flaps (venous flaps) are useful reconstructive options, particularly in the repair of defects with segmental vessel loss. They are relatively easy to harvest and confer several benefits at the donor site. However, given that they are based on a single central vein, their survival is notoriously unreliable and they are susceptible to ischemia and venous congestion. Various designs have been suggested to improve the circulatory physiology, and hence survival, of venous flap. More recent designs involve adaptations to the arrangement and number of efferent veins draining arterialized venous flaps. The most commonly used classification

system for venous flaps, proposed by Chen, Tang, and Noordhoff, does not afford adequate description of these alternate designs. This article offers a classification system that can incorporate all reported modifications to venous flaps. This simple adaptation to the classification system proposed by Chen et al. restores its usefulness in describing modern variations to venous flap design. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012. “
“When reconstructing combined defects of the cervical spine and the posterior pharyngeal wall

the goals are bone stability along with continuity of the aerodigestive tract. We present a case of a patient with a cervical spine high throughput screening compounds defect, including C1 to C3, associated with a posterior pharyngeal wall defect after excision of a chordoma and postoperative radiotherapy. The situation was successfully solved with a free fibula osteo-adipofascial flap. The reconstruction with a fibula osteo-adipofascial flap provided several benefits Methocarbamol in comparison with a fibula osteo-cutaneous flap in our case, including an easier insetting of the soft tissue component at the pharyngeal level and less bulkiness of the flap allowing our patient to resume normal deglutition. © 2013 Wiley Periodicals, Inc. Microsurgery 34:314–318, 2014. “
“The objective of this preliminary study was to develop a reabsorbable vascular patch that did not require in vitro cell or biochemical preconditioning for vascular wall repair. Patches were composed only of hyaluronic acid (HA). Twenty male Wistar rats weighing 250–350 g were used. The abdominal aorta was exposed and isolated. A rectangular breach (1 mm × 5 mm) was made on vessel wall and arterial defect was repaired with HA made patch. Performance was assessed at 1, 2, 4, 8, and 16 weeks after surgery by histology and immunohistochemistry. Extracellular matrix components were evaluated by molecular biological methods.