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We observed a statistically significant lower risk for distal colorectal adenoma among regular aspirin users com pared with nonregular users (multivariate-adjusted relative risk, 0.75 [95% CI, 0.66 to 0.84]). The effect was similar for both small (_1 cm in diameter) and large (_1 cm in diameter) adenomas and adenomas with tubular or villous histologic characteristics. Our results also remained unchanged when we evaluated the risk for new or incident adenoma on follow-up endoscopy in the subcohort of participants who were known to be adenoma free on a previous examination. We considered the possibility that differential rates of gastrointestinal bleeding might have influenced the observed findings. However, when we examined women who underwent endoscopy only for asymptomatic screening as well as those who underwent evaluation of occult or visible bleeding, we found a similar inverse association for regular aspirin use in each stratum. To assess long-term, consistent aspirin use more accurately, we focused our analyses on women who reported regular aspirin use on 2 and 3 consecutive biennial questionnaires before endoscopy and compared their incidence with that of women who were nonusers on 2 and 3 consecutive questionnaires, respectively. However, when adenoma risk was compared according to regular aspirin use reported on 1, 2, and 3 consecutive questionnaires, there did not seem to be any greater benefit among more consistent users. The apparent benefit associated with aspirin use was substantially greater with increasing dose. Compared with participants who took no aspirin, women who used the equivalent of 0.5 to 1.5 standard aspirin tablets per week experienced a modestly lower risk for adenoma (multivariate-adjusted relative risk, 0.80 [CI, 0.70 to 0.93]), whereas women who used more than 14 tablets per week experienced the greatest risk reduction (multivariateadjusted relative risk, 0.49 [CI, 0.36 to 0.65]; P _ 0.001 for trend). Since women who reported higher doses of aspirin use may have used the drug for longer periods, we further examined the influence of aspirin dose after additionally controlling for years of aspirin use. However, even after adjustment for duration of use, the effect of increasing aspirin dose remained strong (P _ 0.001 for trend).

We considered the possibility that the influence of aspirin dose was due to more consistent long-term aspirin use among women taking higher doses. Therefore, we repeated our analysis after restricting the cohort to the 11 857 participants who reported consistent aspirin use on 3 consecutive questionnaires before endoscopy and those who reported no use on 3 consecutive questionnaires. Among women who reported consistent aspirin use over 6 years, we continued to observe a statistically significant reduction in adenoma risk with increasing aspirin dose (P _ 0.001 for trend). Again, when adjustments were made for duration of use, increasing aspirin dose remained strongly predictive of lower adenoma risk (P _ 0.006 for trend).

Women were grouped according to categories of aspirin use, as computed from the questionnaire completed before diagnosis of an adenoma or before negative results on endoscopy. For the primary analyses, as a measure of association, we used relative risks, defined as incidence of adenoma among regular aspirin users divided by the corresponding incidence among participants who were nonregular users. Logistic regression models were used to adjust for age, according to 5-year categories, and multiple known or suspected adenoma risk factors simultaneously. We used odds ratios from these models to approximate relative risk. Because adenomas are typically asymptomatic and may have been present for some time, we also examined the risk for incident adenomas by restricting our analysis to women who had undergone a previous endoscopy more than 2 years before their most recent endoscopy and had been determined to be adenoma free. We also conducted stratified analyses to determine whether aspirin intake was modified by the indication for endoscopy or other risk factors. To assess long-term, consistent use more accurately, we focused our analyses on women who reported regular aspirin use on 2 and 3 consecutive biennial questionnaires before endoscopy and compared their incidence to women who were nonusers on 2 and 3 consecutive questionnaires. We calculated relative risks according to dosage of aspirin based on the number of standard tablets used per week. On the basis of our earlier analysis, we also examined relative risks according to duration of aspirin use by using the number of years of use reported in 1980 with updating of this variable every 2 years. We performed tests for interaction by entering into the multivariable model the cross-product term of aspirin dose and duration of aspirin use (each modeled continuously). We used the SAS statistical package, version 8.2 (SAS Institute, Cary, North Carolina) for all analyses. All P values are 2-sided.

Role of the Funding Source
The funding source had no role in the design and conduct of the study; the collection, analysis, or interpretation of the data; or the decision to submit the manuscript for publication.

RESULTS
Among the 27 077 eligible women, we documented 1368 pathologically confirmed cases of adenoma of the distal colon and rectum. Within this cohort, 38% were regular aspirin users, defined as taking 2 or more standard aspirin tablets per week. Compared with participants who did not report aspirin use, women reporting the highest levels of use were less likely to exercise regularly and were more likely to have a family history of colorectal cancer; to take multivitamins; and to undergo endoscopy for bleeding, abdominal pain, or change in bowel habits. In addition, women who reported more frequent aspirin use consumed more alcohol and folate.

We assessed intake of aspirin in 1980, 1982, 1984, 1988, 1990, 1992, 1994, and 1996. In 1980, we asked women whether they used aspirin in most weeks and, if the answer was yes, the number of pills or capsules taken each week and the number of years of use. In 1982, we inquired whether they currently took aspirin at least once per week did not consistently assess intake of nonsteroidal anti-inflammatory drugs until 1990; in 1990 and 1992, women were asked the number of days per month of anti-inflammatory drug use (for example, ibuprofen, Naprosyn [Roche, Nutley, New Jersey], and Advil [Whitehall Robins Healthcare, Madison, New Jersey]) (none, 1 to 4, 5 to 14, 15 to 21, or _22). In 1994 and 1996, women were also asked to indicate “yes” if they used other anti-inflammatory medications (for example, Advil, Motrin [McNeil, Fort Washington, Pennsylvania], and Indocin [Merck, West Point, Pennsylvania]). The reasons for aspirin use were not assessed for the entire cohort, but a questionnaire was sent in 1990 to a sample of 100 women who reported taking 1 to 6 aspirin tablets per week (90% response) and 100 women who reported taking 7 or more aspirin tablets per week (92% response) on the 1980, 1982, or 1984 questionnaire. The major reasons for use among women taking 1 to 6 aspirin tablets and 7 or more aspirin tablets per week were headache (32% and 18%, respectively), arthritis and other musculoskeletal pain (30% and 50%, respectively), a combination of headache and musculoskeletal pain (16% and 15%, respectively), cardiovascular disease prevention (9% and 8%, respectively), and other reasons (13% and 9%, respectively).

Ascertainment of Cases
On each questionnaire, we inquired whether participants underwent sigmoidoscopy or colonoscopy; the indications for the procedure; whether colon or rectal polyps had been diagnosed; and, if so, the date of diagnosis. When a participant reported a diagnosis, we obtained her informed consent to acquire medical records and pathology reports. Study physicians, blinded to exposure data, reviewed all records and extracted data on histologic type, anatomic location, and size of polyps. We did not ask participants to specify either sigmoidoscopy or colonoscopy. On the basis of secular trends, there was probably a gradual shift in the relative proportion of colonoscopies compared with sigmoidoscopies. However, we assumed that a substantial portion of all procedures were sigmoidoscopies, which encompass only examination of the distal colon and rectum. As in previous studies, to avoid detection bias, in all main analyses we defined cases as 1 or more pathology-verified adenomas less than 60 cm from the anus. If more than 1 adenoma was diagnosed, the case was classified by the largest size and most advanced histologic characteristics. We classified women with adenomas proximal to the descending colon but without synchronous distal adenoma as non–case participants. We also performed a secondary analysis defining these proximal adenomas as the outcome of interest to examine the influence of aspirin on the risk for proximal adenomas.

In 1984 and 1988, we queried the average number of days per month of aspirin use (none, 1 to 4, 5 to 14, 15 to 21, or _22) and the number of aspirin tablets usually taken (1, 2, 3 to 4, 5 to 6, or _7). In 1990 and 1992, women were asked the number of days per month of aspirin use (none, 1 to 4, 5 to 14, 15 to 21, or _22), and in 1994 and 1996, women were asked the frequency of aspirin use (_1 day per month, 1 to 3 days per month, 1 to 2 days per week, 3 to 4 days per week, 5 to 6 days per week, or daily) and number of aspirin tablets per week (0, 0.5 to 2, 3 to 5, 6 to 14, or _15). Early in the study, most women used standard-dose aspirin tablets; however, to reflect overall secular trends in consumption of low-dose or baby aspirin, the 1994 and 1996 questionnaires asked participants to convert intake of 4 baby aspirin to 1 adult tablet. As previously described, some regrouping of responses was required to adjust for the differing ways in which habits of aspirin use were recorded.

On the basis of previous analysis of this cohort, women who reported taking 2 or more standard aspirin tablets per week were defined as regular users, whereas those who reported less aspirin use were defined as nonregular users. We patterns of aspirin use, and examination by either colonoscopy or sigmoidoscopy (not specified), as well as indications for the procedure. For our study sample, we excluded women who did not complete the baseline dietary questionnaire, did not provide data on aspirin use before endoscopy, or reported implausible dietary (for example, _600 calories/d) or aspirin information (for example, responded “yes” to use, but then recorded zero aspirin tablets per week). In addition, we excluded women who reported a baseline history of cancer (except nonmelanoma skin cancer), adenoma, inflammatory bowel disease, or a familial polyposis syndrome. To minimize detection and selection bias in the analysis of adenomatous polyps, we restricted the cohort to women who reported undergoing either colonoscopy or sigmoidoscopy during the study period. Through 31 May 1998, after these exclusions, 27 077 women were eligible for analysis. Compared with women who did not undergo endoscopy (n _ 50 273), the women who underwent endoscopy during the study period (n _ 27 077) were more likely to have a family history of colorectal cancer, use postmenopausal hormones currently or in the past, consume multivitamins regularly, and not smoke (data not shown). All participants provided written informed consent. The Human Research Committee at the Brigham and Women’s Hospital approved this study.

Considerable experimental, epidemiologic, and clinical trial evidence suggests that aspirin reduces the risk for colorectal adenoma and cancer. Previous studies demonstrate that a statistically significant reduction in colorectal cancer risk probably requires at least a decade of regular aspirin use, whereas recent randomized trials demonstrate that short-term use of aspirin can prevent recurrent adenomas. Nonetheless, the duration needed for the primary prevention of colorectal adenoma has not been clearly defined. Moreover, for both cancer and adenoma prevention, the optimal chemopreventive dose of aspirin remains unknown. Randomized intervention trials of the effect of aspirin on the rate of adenomas have offered compelling evidence of causality but have provided only limited and conflicting data on the dose–response relationship and the necessary duration of therapy. In a randomized, placebo-controlled trial of 22 071 male physicians, low-dose aspirin (325 mg of aspirin every other day) was not associated with a statistically significant decrease in colorectal adenomas during the first 5 years of follow-up. In contrast, recent results from a trial of patients randomly assigned after endoscopic polypectomy suggested that low-dose aspirin (81 mg/d) reduced adenoma recurrence by 19% at 3 years, whereas standard-dose aspirin (325 mg/d) had no effect. Furthermore, it remains unclear whether a benefit to aspirin use can be generalized to a low-risk population without a history of adenoma. In patients with familial polyposis, intervention trials of the nonsteroidal anti-inflammatory drug sulindac demonstrated a benefit in secondary prevention but not primary prevention of adenoma. We prospectively examined the relation between aspirin and colorectal adenoma in a large cohort of women enrolled in the Nurses’ Health Study. This cohort, which provides detailed and updated information on aspirin use across a wide range of intake, permitted a more comprehensive examination of the effect of duration and dose on the primary prevention of sporadic colorectal adenomas than would be feasible in a placebo-controlled trial.

METHODS
Study Sample
The Nurses’ Health Study began in 1976 when 121 701 U.S. female registered nurses 30 to 55 years of age completed a questionnaire on risk factors for cancer and coronary heart disease. With a follow-up rate of more than 92%, every 2 years we have mailed questionnaires to update information and identify newly diagnosed cases of cancer and other diseases. In 1980, the questionnaire was expanded to include a validated assessment of diet, and, if yes, how many aspirin tablets per week (1 to 3, 4 to 6, 7 to 14, or _15).

In this cluster randomized crossover trial, routine sterile gloving just before venipuncture reduced blood culture contamination rates by approximately 50%. To the best of our knowledge, our study is the first to evaluate the influence of sterile gloving on blood culture contamination rates. Although sterile gloving is a basic aspect of aseptic technique, most previous studies did not consider the gloving method when they evaluated blood culture contamination rates. To minimize confounding caused by a difference in phlebotomy skills and the consequential contamination risk for individual interns, we used a crossover design and included a random effect of interns for the statistical model.

Previous studies found that trained phlebotomy teams decrease blood culture contamination rates compared with resident physicians or nurses. These findings suggest that personal phlebotomy skills influence blood culture contamination rates. Our data also showed that the contamination rates were diverse according to individual interns. Although blood culture was done by interns rather than dedicated phlebotomists in this study, the baseline contamination rate was relatively low, even when possible contaminants were included; although the baseline contamination rates reported by previous randomized, controlled trials were 3% to 9%, our contamination rate was roughly 1% during optional sterile gloving. The exclusion of the emergency department and pediatric ward may partly explain our low contamination rates because contamination rates tend to be higher in these areas than elsewhere. In addition, comprehensive education on the standard protocol for specimen collection might have contributed to the low contamination rates. Furthermore, an awareness of the research might increase intern adherence to the standard protocol. Our data imply that adherence to current guidelines can reduce blood culture contamination rates to approximately 1%, as shown in our control period.

The lower blood culture contamination rates associated with routine sterile gloving may indicate the possibility of contamination of the nonsterile gloves worn by the interns. An outbreak of contaminated blood cultures caused by nonsterile gloves contaminated by Bacillus species was reported. However, in our study, the contaminants during optional sterile gloving were diverse and were mainly skin flora, which suggests that an outbreak due to collective contamination of nonsterile gloves was less likely. The difference in blood culture contamination rates between the routine and optional sterile gloving groups was highest in the intensive care unit, in which relatively higher contamination rates during optional sterile gloving may be explained by phlebotomy difficulties due to the poor vascular condition of patients with chronic or severe illness, as well as by the less common use of sterile gloving as self-reported by the interns. A heavy workload in the busy intensive care unit might make optional sterile gloving by interns less common. Some previous studies also reported higher contamination rates in intensive care units than in general wards, although data comparing the contamination rates of intensive care units and other hospitalization units are limited.

The contamination rate based on hospital unit was 1.0% (69 of 7027 cultures) in general wards, 0.4% (11 of 2446 cultures) in hematology wards, and 1.3% (14 of 1047 cultures) in the intensive care unit if possible contaminants (P _ 0.039) were included and 0.8% (56 of 7027 cultures) in general wards, 0.4% (9 of 2446 cultures) in hematology wards, and 0.6% (6 of 1047 cultures) in the intensive care unit if only likely contaminants were included (P _ 0.107). The contamination rate based on gloving method sequence was 1.0% (54 of 5397 cultures) in routine-tooptional sterile gloving and 0.8% (40 of 5123 cultures) in optional-to-routine sterile gloving if possible contaminants were included (P _ 0.30). The contamination rate was 0.7% (40 of 5397 cultures) in routine-to-optional sterile gloving and 0.6% (31 of 5123 cultures) in optional-toroutine sterile gloving if only likely contaminants were in cluded (P _ 0.40). The mean contamination rates by interns were 1.0% (SD, 1.0%; interquartile range, 0% to 1.5%) if possible contaminants were included and 1.0% (SD, 0.7%; interquartile range, 0% to 1.1%) if only likely contaminants were included. Generalized mixed models demonstrated significant differences in contamination rates between routine and optional sterile gloving, regardless of the classification into contaminants or pathogens. When possible contaminants were included, the contamination rate was 0.6% in routine sterile gloving and 1.1% in optional sterile gloving (adjusted odds ratio, 0.57 [95% CI, 0.37 to 0.87]; P _ 0.009). If only likely contaminants were included, the contamination rate was 0.5% in routine sterile gloving and 0.9% in optional sterile gloving (adjusted odds ratio, 0.51 [CI, 0.31 to 0.83]; P _ 0.007).
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Adherence to Gloving Methods
The interns reported the actual gloving methods for 8082 (76.8%) of 10 520 blood cultures. The reporting rate for the actual gloving methods used was 76.3% (5363 of 7027 cultures) in general wards, 80.5% (1968 of 2446 cultures) in hematology wards, and 71.7% (751 of 1047 cultures) in the intensive care unit (P _ 0.133). The reporting rate was 76.8% (4045 of 5265 cultures) in routine sterile gloving and 76.8% (4037 of 5255 cultures) in optional sterile gloving (P _ 0.54). No statistically significant difference was found in contamination rates between blood cultures obtained with or without known gloving methods (0.8% vs. 0.9% [P _ 0.39] if possible contaminants were included and 0.7% vs. 0.7% [P _ 0.88] if only likely contaminants were included).

During routine sterile gloving, 3791 (93.7%) of 4045 blood cultures were done wearing sterile gloves, whereas in optional sterile gloving, 296 (7.3%) of 4037 blood cultures were carried out with sterile gloving. The adherence rate to sterile gloving during the routine gloving period was 92.3% (1973 of 2138) in the routine-to-optional sterile gloving group and 95.3% (1818 of 1907) in the optionalto- routine sterile gloving group (P _ 0.68). In optional sterile gloving, sterile gloves were worn for 2.8% of blood draws (55 of 1977) in the routine-to-optional sterile gloving group and 11.7% (241 of 2060) in the optional-toroutine sterile gloving group (P _ 0.001). In routine sterile gloving, the adherence rate to sterile gloving was 93.9% (2520 of 2685) in general wards, 92.8% (925 of 997) in hematology wards, and 95.3% (346 of 363) in the intensive care unit (P _ 0.006). In optional sterile gloving, sterile gloves were worn for 7.8% (209 of 2678) of blood draws in general wards, 8.0% (78 of 971) in hematology wards, and 2.3% (9 of 388) in the intensive care unit (P _ 0.001).

In response to B. burgdorferi, in vitro and in vivo transcription and expression of MMP-9, but not its posttranslational activation, occurred in a CD14-independent manner. In contrast, Zhao et al24 demonstrated a requirement for CD14 in both the transcription and expression of MMP-9 in human macrophages and fibroblasts. Left unanswered because of differences in experimental design is whether CD14 has a species-specific role in posttranslational activation of MMP-9. One critical distinction between the two studies is that Zhao et al relied entirely on in vitro experimentation with immortalized human monocytic and fibroblastic cell lines (ie, U937 and ATCC SCRC-1042.2, respectively), whereas, in the present study, the role of CD14 in regulation of MMP-9 at the transcriptional, translational, and posttranslational levels was evaluated during natural infection, allowing for more complex interactions among the pathogen, a variety of host cell types, and the ECM.

Finally, a phenomenon indicative of fibrosis reported herein was greater collagen staining in the joints of infected CD14−/− versus CD14+/+ mice. Inflammation and fibrosis are two interrelated processes with intertwined signaling events, especially when elicited by infectious agents. As part of the tissue repair process, neutrophils and macrophages coordinate their efforts to clear residual bacterial components and effect removal of apoptotic and necrotic cellular debris from the site of infection. Chemokines, cytokines, and growth factors released by these phagocytes recruit lymphocytes to and activate fibroblasts within the inflammatory focus. In addition, the differentiation of macrophages into fibroblast-like cells called myofibroblasts occurs within these foci. Fibroblasts and myofibroblasts participate in the reconstruction of ECM by virtue of their ability to deposit collagen and secrete MMPs. Tissue repair, a highly regulated process, is subject to dysregulation in the prolonged presence of bacteria and cytokines. Perpetual TLR stimulation at sites of infection or noninfectious injury can amplify profibrotic signaling and engender fibrotic disorders. In renal injury and cystic fibrosis, excessive TLR2 signaling induces several fibrotic genes. The absence of CD14 on macrophages results in a 50-fold increase in B. burgdorferi–stimulated transcription of tlr2 and protracted surface expression of protein, which, in turn, is partly responsible for greater production of proinflammatory cytokines. The ability of macrophages to trigger apoptosis in myofibroblasts also may depend on CD14 expression. Thus, in its absence, extended survival of myofibroblasts and their accumulation within joints could contribute to greater collagen deposition in CD14−/− mice. Alternatively, the inability of spirochetes to induce inducible nitric oxide synthase in CD14−/− macrophages may be a factor because the absence of inducible nitric oxide synthase activity has been associated with greater transcription of collagen genes and higher collagen deposition.49 Although not significant, a trend toward higher transcription of collagen genes was observed in CD14−/− joints compared with those of wild-type mice. Considering the potential implications of greater deposition and/or diminished turnover of ECM, it is important to note that B. burgdorferi express decorin-binding proteins A and B, especially when they reside in the skin and joints where collagen content is high. Surface expression of decorin-binding proteins A and B anchors spirochetes to collagen and may facilitate their persistence in mouse and human joints. The ability of spirochetes to embed themselves in dense collagen bundles may offer protection from both antibodies and antibiotics. Putting all this information together, we can speculate that CD14 not only helps in the activation of MMP-9 necessary for degradation of existing collagen but also may limit the continued production of collagen. Control of these fibrotic processes by CD14 not only leads to higher neutrophil recruitment and clearance of spirochetes but also deprives B. burgdorferi of “hide-outs” within joints.

This study describes a previously unappreciated function for CD14 in bacterial clearance by regulating the extravasation of neutrophils into the joints of B. burgdorferi–infected mice. Although CD14 deficiency leaves unaltered the production of CXCL neutrophil chemoattractants and transcription of mmp-9 both in vitro and in vivo, a significant decrease in the pool of active MMP-9 occurs. The cascade effect of this reduction in MMP-9 activity was diminished turnover of ECM and generation of PGP, limited recruitment of neutrophils into the joint space, and impaired clearance of spirochetes. These findings raise the intriguing question of whether individual variation in the efficiency of neutrophil-mediated clearance of B. burgdorferi might underlie differences in the severity and/or duration of Lyme arthritis observed in the patient population. If such differences are responsible for the range of clinical presentation observed, it suggests avenues for the development of adjunctive therapy designed to augment host immunity, perhaps through small molecule–mediated enhancement of neutrophil recruitment.

As the chemokine gradient wanes, the influx of neutrophils diminishes; however, in several inflammatory conditions where tissue injury is a dominant feature, neutrophils continue to enter inflamed tissues even in the absence of detectable levels of chemokines. Prolongation of neutrophil migration results from generation of collagen fragments released through turnover of the ECM. Weathington et al have demonstrated the neutrophil chemotactic potential of small tripeptide fragments of PGP generated by the action of MMP-9. The capacity of MMP-9 to regulate neutrophil recruitment and thus influence the intensity and duration of disease is well documented.35 It is worth noting that correlation between MMP-9 activity and disease severity can vary and depends on whether the disease is infectious or noninfectious in origin.

For example, the severity of rheumatoid arthritis correlates positively with the level of active MMP-9 and the number of neutrophils recruited into the joint space. In infectious diseases, because neutrophils play a role in both tissue damage and clearance of pathogens, the balance between the two becomes delicate and essential to maintain. MMP-9 deficiency increases the survival of F. tularensis–infected mice by avoiding excessive neutrophil recruitment and greater tissue damage in the lung. However, at the other extreme, complete antibody-mediated depletion of neutrophils increases mortality after challenge of mice with a sublethal dose of Francisella. In a sepsis model, where MMP-9−/− mice were challenged with Escherichia coli, animals showed severe impairment of neutrophil recruitment in late-phase disease and higher bacterial burden. Staphylococcus aureus–triggered septic arthritis also is exacerbated by MMP-9 deficiency wherein mice display a higher frequency and severity of arthritis and harbor increased numbers of bacteria in their joints. Similarly, as Lyme arthritis develops there is a tug-of-war between clearance of spirochetes (a necessary prelude to resolution of disease) and damage to tissues. Herein, we observed lower active MMP-9 in the joints of CD14−/− mice, which was associated with greater maintenance of collagen integrity and a trend toward lower levels of PGP. Consistent with this observation were reduced numbers of neutrophils (especially at the height of the arthritic reaction) and impaired clearance of bacteria. MMP-9−/− mice showed a similar phenotype, including maintenance of collagen integrity, fewer recruited neutrophils, and a higher spirochetal burden in joints at 6 weeks after initial infection. Recently, Heilpern et al42 evaluated the role of MMP-9 in dissemination of B. burgdorferi and reported no differences in the rate and/or pattern of spirochetal dissemination, bacterial burden, cytokine/chemokine production, or the extent and/or composition of cellular infiltrates (including neutrophils). However, they found a lesser degree of joint swelling in the absence of MMP-9, the opposite of what we observed (data not shown). Discordance among the findings of Heilpern et al, the results presented herein, and our current understanding of the role of MMP-9 in mediating neutrophil recruitment may be due to differences in the genetic background of mice used (C3H/HeN in the study by Heilpern et al and FVB/N in the present study).

MMP-9 Deficiency Leads to Lower Recruitment of Neutrophils and Impaired Clearance of Borrelia from Joints

Finally, the extent of neutrophil recruitment into the joints of MMP-9+/+ and MMP-9−/− mice was evaluated. As was observed in CD14−/− joints, the joints of B. burgdorferi–infected mice lacking MMP-9 have significantly fewer neutrophils than MMP-9+/+ animals as evidenced by the amount of MPO staining at 3 (data not shown) and 6 weeks p.i. Data compiled from two independent experiments show a nearly 75% reduction in neutrophil recruitment in the absence of MMP-9. To validate our overall hypothesis that diminished MMP-9 activity results in lower neutrophil recruitment and impaired bacterial clearance from the joint, we evaluated bacterial burden in different organs of infected MMP-9+/+ and MMP-9−/− mice. After infection of mice with 1 × 105 B. burgdorferi, genomic DNA was isolated from the heart, joint, ear, and bladder at 1, 3, and 6 weeks p.i., and qPCR was used to quantify bacterial burden. Both the genotypes of mouse were equally susceptible to infection and harbored similar numbers of bacteria in their organs at 1 and 3 weeks p.i. However, by 6 weeks p.i. the bacterial burden was markedly greater in MMP-9−/− joints than in those of wild-type mice (P < 0.01). Discussion

During transmission of B. burgdorferi to the mammalian host, neutrophils are recruited to the tick feeding site. However, because of the neutrophil-inhibitory effects of I. scapularis saliva, not all spirochetes are cleared from the site of inoculation. Subsequent dissemination to distal sites away from the effects of tick-salivary proteins allows for efficient phagocytosis and killing of B. burgdorferi by neutrophils. This scenario is supported by the finding that Borrelia infection of mice wherein neutrophils were depleted using anti–GR-1 antibody results in higher bacterial burden and early onset of arthritis.4 Similarly, when B. burgdorferi genetically engineered to secrete KC were syringe inoculated into mice, the responding neutrophils effectively cleared spirochetes, thus short-circuiting the infectious process.6 This effect was reversed when Borrelia-expressed KC was neutralized by anti-KC antibodies.

CD14 facilitates interactions between bacterial products and the TLR signaling machinery, which promote innate immune responses. Nevertheless, its absence in mice and from monocytes/macrophages results in hyperresponsiveness to live B. burgdorferi, which leads to greater production of proinflammatory cytokines. One of the more paradoxical consequences of CD14 deficiency is that despite elevated levels of cytokines with potent antimicrobial activities, CD14−/− mice harbor 10-fold more bacteria in their joints than do wild-type animals. This led us to examine whether CD14 signaling regulates the recruitment of neutrophils in a model of Lyme borreliosis. Traditionally, neutrophil recruitment is regulated through the establishment of a chemotactic gradient formed by the release of chemokines at the site of infection or noninfectious tissue injury. On recruitment, neutrophils release their granular contents into the extracellular milieu as a means to kill bacteria, remodel the ECM through the action of degradative enzymes, and recruit other leukocytes, such as macrophages. Neutrophils are short-lived cells; they die at the site of infection and then are cleared by the incoming macrophages and DCs.

In addition to potential regulation at the transcriptional, posttranscriptional, and translational levels, the action of MMP-9 can be controlled at the posttranslational level as well. As such, we looked at the activation status of MMP-9 by gelatin zymography with the rationale that perhaps CD14 regulates the conversion of MMP-9 from its pro- form to its active form. The upper band corresponding to inactive MMP-9 was abundant in both genotypes and did not appear to increase significantly on infection of animals with B. burgdorferi. In contrast, active MMP-9 levels were elevated on infection and were higher in the joints of CD14+/+ than in CD14−/− mice at 3 and 6 weeks p.i.

Differences in CD14-Dependent MMP-9 Activity Are Reflected in the Extent of Collagen Turnover

Because active MMP-9 levels were lower in B. burgdorferi–infected CD14−/− joints, one would predict the integrity of collagen to be greater than in wild-type mice. To test this hypothesis, collagen within Borrelia-infected joints was visualized with Masson’s trichrome stain. In the absence of CD14, collagen structure within the joint space is far more intact (as evidenced by dark blue staining) than in the joints of CD14+/+ mice. To quantify differences between the genotypes, blue color extraction using Adobe Photoshop was performed as previously described. Joints of CD14−/− mice were found to have nearly twice as much collagen staining compared with their wild-type counterparts. In addition, total protein preparations from isolated joints were assayed by mass spectrometry for the presence of acetylated PGP, which is generated by the degradation of collagen through the action of MMP-9. The levels of PGP in the joints of infected mice were significantly higher than those of uninfected control animals (P = 0.00014), and although not significantly different, there was a trend toward more PGP in the joints of CD14+/+ mice than in mice lacking CD14 (8.497 ± 0.6206 versus 7.215 ± 1.008). Finally, given prior evidence of dysregulation of gene transcription in the absence of CD14, we determined whether differences in collagen content between wild-type and CD14−/− joints might also reflect greater transcription of collagen genes in the latter, where hyperinflammatory conditions exist. To explore this possibility, we measured the transcript levels of 10 different collagen genes at 3 weeks after inoculation. Two genes, col2α1 and col10α1, were transcribed in the absence of CD14 to a greater, although not statistically significant, extent (data not shown).

B. burgdorferi–Infected Joints from MMP-9−/− Mice Have Decreased Collagen Turnover

In the absence of CD14, we observed decreased MMP-9 activity and a corresponding maintenance of collagen integrity. To test directly whether differences in CD14-regulated MMP-9 activity were responsible, collagen integrity was evaluated in the Borrelia-infected joints of FVB/N mice and their MMP-9–deficient (MMP-9−/−) counterparts. In the absence of MMP-9, the integrity of collagen was maintained to a higher degree than in wild-type mice with approximately four fold greater collagen content in MMP-9−/− mice than in those animals expressing MMP-9.