A hallmark cytokine associated with tumor-induced immunosuppression is TGF-β1. Although we detected increased circulation of TGF-β1 in tumor-bearing animals in some experiments, it did not exert an apparent inhibition on the autoimmune Teff cells at a distal site in healthy tissues. At cellular levels, Treg cells and MDSCs have long been recognized as critical mediators of immunosuppression in cancer. Our studies with self-antigen-specific T cells highlighted an increased

potency of these regulatory mechanisms in tumors versus healthy tissues. The molecular mechanisms responsible for the local immunosuppression remain to be elucidated. Possibly, a suppressive cytokine milieu, directly or indirectly related to Treg cells and MDSCs, inactivates Teff cells at the tumor site, which could be reactivated by an agonistic cytokine stimulation [40] or a global alteration of tumor gene expression profiles [41]. This study implicates CTLA4. Ponatinib research buy Suggestive of the intertwining between autoimmunity and antitumor immunity, protection from cancer is often associated with the same polymorphisms of the CTLA4 locus that are linked to autoimmune susceptibility [15, 18-20]. A conditional knockout model

established an essential role for CTLA4 in Treg cells Cisplatin purchase [8]. Its intrinsic role in Teff cells has also been well-documented [9, 10]. Our study with a CTLA4 shRNA model indicates a distinction between quantitative variation in CTLA4 and the “all-or-nothing” model of CTLA4 knockouts. A subtle reduction of CTLA4 did not impair Treg-cell function, but substantially promoted Teff-cell capacity in tumor settings. An expansion of immunotherapy trials has generated a plethora of novel ideas in cancer immunology. The entangling of auto-immunity toxicity with antitumor benefit has provoked a shift of perspective whereby autoimmune side effects are considered

not only a welcome marker but actual effectors for antitumor immunity [7]. A direct comparison of PIK3C2G cancerous versus healthy tissue in interaction with self-antigen-specific Teff cells revealed their intrinsic potential in tumor eradication. However, they were subjected to regulatory mechanisms that have been evolved to induce tolerance to nonmalignant self-tissue, even more so in the tumor microenvironment. Therefore, self-antigen can be effectively targeted for antitumor immunity, but harnessing the tumor-destruction capacity of self-antigen-specific T cells requires effective strategies to overcome the suppressive microenvironment at the tumor site. CTLA4 blockade therapies can abrogate suppressive tumor milieu by reverting the local predominance of Treg cells over self-antigen-specific Teff cells. On the other hand, a subtle reduction of CTLA4 reflecting genetic variations may substantially alter an immunoprivileged environment evolved in a solid tumor through an intrinsic impact on Teff cells.

HAN IN MEE, RYU HAN JAK, KIM EUN JIN, PARK JUNG TAK, HAN SEUNG HYEOK, YOO TAE-HYUN, KANG SHIN-WOOK, CHOI KYU HUN, OH HYUNG JUNG Department of Internal Medicine, College of Medicine, Yonsei University Introduction: Continuous renal replacement therapy (CRRT) has been widely used in critically ill acute kidney injury (AKI) patients. Some centers consist of a specialized CRRT team (SCT)

with physicians and nurses, but few studies have been yet reported on the superiority of SCT control. Methods: A total of 551 patients, who received CRRT between CT99021 mouse August 2007 and August 2009, divided into two groups based on the controller of CRRT. The impact of the CRRT management was compared between two groups. Results: The 28-day mortality rate was significantly lower in SCT group compared with conventional team approach (CTA) group (P = 0.031). In contrast, the number of used filters, total down-time, down-time per day, ICU length of day in CTA group were significantly higher compared to SCT

group (6.2 vs. 5.0, P = 0.042; 31.2 vs. 22.3 hrs, P < 0.001; 5.0 vs. 3.8 hrs, P < 0.001; 27.5 vs. 21.1 days, P = 0.027, respectively), while filter life-time and effluent UFR in CTA group were significantly lower than SCT group (19.3 vs. 23.1 hrs, P = 0.035; 28.0 vs. 29.5 ml/kg/hr, P = 0.043, respectively). Conclusion: A SCT group might be beneficial for mortality improvement of AKI patients requiring CRRT. GUANG-HUAR YOUNG1, VIN-CENT WU2 1Department of Surgery; 2Department of Internal Medicine, Division of Nephrology, National Taiwan University Hospital, Obeticholic Acid purchase Taipei Introduction: Renal recovery from acute kidney injury (AKI) is often not achieved because of accompany with new injuries during the repair phase. Indoxyl sulfate (IS), a potential vascular toxin retains in AKI patients could significantly activate most of the intra-renal renin–angiotensin system (RAS) components. The inappropriate activation of the RAS contributes to imbalance of ACE/AngII/AT1 axis versus ACE2/Ang1-7/MAS axis after renal injury.

Here we examined renal protective effects of direct rennin inhibitor (DRI) and angiotensin II receptor blockers (ARB) in the IS-mediated AKI. Methods: Human Digestive enzyme proximal tubular epithelial (HK-2) cells were exposed to 1 mM IS and hypoxia (1% oxygen) in the absence or presence of DRI (20 nM Aliskiren) or ARB (200 nM Losartan) for 72 hours. The mice with IS-mediated AKI, induced by unilateral renal ischemia/reperfusion injury and IS (100 mg/kg/day, from day 1 to 3), were randomly divided into 5 groups: the Sham group, the Model group, the Aliskiren group (25 mg/kg/day), the Losartan group (10 mg/kg/day) and the Combination group. Results: Most of the RAS components including angiotensinogen and ACE were activated in HK2 cells under IS and hypoxia condition. In contrast to ACE, ACE2 represent a bidirectional way which is increased during the early stage but decreased near-baseline levels at the later stage (Figure 1).

If pushed to provide a criticism of this book, I would mention that it is sometimes difficult to keep track of the much-used abbreviations, as many of these have been appointed much earlier on in the text. However, this can prove helpful as revision of previously read or ‘skipped’ text in this way can help to reinforce knowledge. With its rich presentation and Osborn’s friendly and authoritative tone throughout,

this book is enjoyable to read and a pleasure to use. I would Dabrafenib cost recommend it highly and feel it is well worth its price. “
“Reinhard B. Dettmeyer . Forensic Histopathology: Fundamentals and Perspectives . Springer-Verlag , Berlin , 2011 . 454 Pages. Price £126.00 (Amazon) (hardcover). ISBN- 10 3642206581 ; ISBN- 13 978-3642206580 This book has been compiled by a German forensic pathologist who has embarked on the difficult task of deciphering not only forensic, but also general histopathology related to the autopsy. Very few books are available which detail the histopathological features seen within tissue following a post mortem examination and this is, therefore, an exciting development. The book is divided into 20 chapters and each details different aspects of forensic histopathology

including drug-induced pathologies, alcohol-related PD-0332991 molecular weight histopathology and of course, forensic neuropathology. The first chapter gives an introduction and highlights the use of post mortem histology with several succinct case studies, one of which shows spinal cord necrosis following intrathecal injection. The next chapter, as with many histopathology texts, gives an overview of staining techniques including immunohistochemistry. This chapter is rather brief and to the point but is similar in style

to comparable texts. The author does, however, direct the reader to more specialist texts, if they so desire. There is, however, a very good table detailing some of the more common stains, which trainee pathologists in particular may find a useful reference. Pembrolizumab The book then details histopathology in the setting of trauma and trauma-related deaths followed by drug abuse. Such deaths can often be encountered in the setting of neuropathology, and, therefore, this book serves well to inform the pathologist of features which may be seen in other organs, outwith the nervous system. Neuropathologists specializing in forensic work, or indeed those involved routinely in traumatic deaths, will find this book of immense use. A very good chapter has been compiled on wound age in the case of tissue injuries, and the table which is included giving an outline of dating of fractures will be of particular use. A large component of the book is dedicated to cardiovascular deaths.

, 2004; Mulvey et al., 2005; David et al., 2008; Van De Griend et al., 2009). Recent studies show that USA400 can account for over 98% of MRSA infections in northern Canada (Golding et al., 2011) and has been implicated in isolated PXD101 datasheet MRSA disease in southern Europe (Vignaroli, 2009; Neocleous et al., 2010). However, about 10 years ago, a new source of CA-MRSA arose from one of the ‘traditional’ virulent CCs, CC8. Descending from a USA500

clone through acquisition of various MGEs (Robinson & Enright, 2003; Li et al., 2009), USA300 became the dominant CA-MRSA clone in US (Moran et al., 2006; Hulten et al., 2010; Talan et al., 2011), effectively replacing USA400 clones in most regions (Como-Sabetti et al., 2009; Simor et al., 2010), and has also been isolated from patients in Canada and Mexico (Nichol Talazoparib cost et al., 2011; Velazquez-Meza et al., 2011).

The explosion of USA300 CA-MRSA across North America resulted from a very recent clonal expansion of a successful CA-MRSA clone as demonstrated by very low sequence divergence among geographically distinct USA300 isolates (Kennedy et al., 2008). Given the occurrence of multiple CA-MRSA clones in the population, a formal definition was put forth by the Center for Disease Control and Prevention for CA-MRSA disease as that which is contracted within 48 h of hospital admission by patients not having recently undergone surgery, hemodialysis, prolonged hospitalization, Neratinib manufacturer catheterization, or MRSA colonization (Morrison et al., 2006). Currently in the US, MRSA disease fitting these criteria is almost always caused by USA300 clones, followed by USA400 and occasionally USA1000 and USA1100 (Talan et al., 2011). To complicate matters further, USA300 clones have recently been implicated in causing significant HA-MRSA disease (Popovich et al., 2008; Jenkins et al., 2009; Moore et al., 2009; Hulten et al., 2010), blurring the lines between the two disease

onset environments (Popovich et al., 2008; Jenkins et al., 2009; Moore et al., 2009; Hulten et al., 2010). In some studies, USA300 accounted for at least half of hospital-acquired MRSA infections (Popovich et al., 2008; Hulten et al., 2010). Thus, USA300 represents a highly successful S. aureus clone that emerged in the community and quickly spread throughout the North American continent to become the leading cause of MRSA infection even in healthcare settings. For now, USA300 seems to be primarily limited to North America, while in Europe, South America and Asia, CA-MRSA disease is dominated by divergent clones unrelated to CC8 (e.g. ST30, ST80 and ST59) (Deleo et al., 2010). Given the rapid and efficient transmissibility of USA300 in North America (Pan et al., 2005), it remains to be seen whether these clones will become the dominant source of MRSA disease worldwide. Animal models of S.

In cases with diffuse traumatic axonal injury the microglial reaction is particularly pronounced in the white matter. These results demonstrate that prolonged microglial activation is a feature of traumatic brain injury, but that the neuroinflammatory response returns to control levels after several years. “
“Cerebral selleck kinase inhibitor amyloid angiopathy (CAA) predisposes to symptomatic intracerebral hemorrhage (sICH) after combined thrombolytic and anticoagulant treatment of acute myocardial infarction. However, the role

of CAA in stroke thrombolysis has not been established. Here, we describe a confirmed case of CAA-related hemorrhage in a patient receiving thrombolysis for acute ischemic stroke. On autopsy, immunohistochemistry revealed amyloid-β positive staining in thickened cortical and meningeal arteries at sites of hemorrhage. Further research is urgently needed to determine

the hemorrhage risk related to CAA in stroke thrombolysis and develop better diagnostic tools to identify CAA in the emergency room. “
“Sphingosine-1-phosphate receptor (S1PR) modulating therapies are currently in the clinic or undergoing investigation for multiple sclerosis (MS) STA-9090 datasheet treatment. However, the expression of S1PRs is still unclear in the central nervous system under normal conditions and during neuroinflammation. Using immunohistochemistry we examined tissues from both grey and white matter MS lesions for sphingosine-1-phosphate receptor 1 (S1P1) and 5 (S1P5) expression. Tissues from Alzheimer’s disease (AD) cases were also examined. S1P1 expression was restricted to astrocytes and endothelial cells in control tissues and a decrease in endothelial cell expression was found in white matter MS lesions. In grey matter MS lesions, astrocyte expression was lost in active lesions, while in quiescent lesions it was restored to normal expression levels. CNPase Thalidomide colocalization studies demonstrated S1P5

expression on myelin and both were reduced in demyelinated lesions. In AD tissues we found no difference in S1P1 expression. These data demonstrate a differential modulation of S1PRs in MS lesions, which may have an impact on S1PR-directed therapies. “
“C. Billingham, M. R. Powell, K. A. Jenner, D. A. Johnston, M. Gatherer, J. A. R. Nicoll and D. Boche (2013) Neuropathology and Applied Neurobiology39, 243–255 Rat astrocytic tumour cells are associated with an anti-inflammatory microglial phenotype in an organotypic model Aim: Microglia form a high proportion of cells in glial tumours but their role in supporting or inhibiting tumour growth is unclear. Here we describe the establishment of an in vitro model to investigate their role in astrocytomas. Methods: Rat hippocampal slices were prepared and, after 7 days to allow microglia to become quiescent, rat C6 astrocytic tumour cells were added.

Consistent with the findings of others, Dr. Eisenbarth and colleagues determined that

the Nalp3 inflammasome is important in the adjuvant activity of alum, but that Nalp3 activation is not a universal requirement of Th2 responses 29–31. Although these findings demonstrate that the innate inflammasome pathway can direct an adaptive Th2 immune response, it is not clear that this same inflammasome pathway regulates all Th2 responses or has a role in atopic disease. Thus far, data regarding the role of any inflammasome in mast cell function are limited; however, it is clear that the inflammasome and NLR in general have unique roles in the activation of both the innate and adaptive immune responses. Recent studies have evaluated the immune potentiating Bortezomib abilities

of mast cell activators to enhance vaccine-induced immune responses. Mast cells recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Soman Abraham (Durham, NC) and colleagues recently demonstrated that subcutaneous or nasal administration of small-molecule mast cell activators with vaccine Ags evokes large increases in Ag-specific serum IgG responses 32. These responses were mast cell dependent and correlated with increased DC and lymphocyte recruitment to draining lymph nodes 33. Nasal instillation of these formulations also increased Ag-specific secretory IgA and provided protection against anthrax Silmitasertib supplier lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define

the mast cell as an integral sensory arm of the adaptive immune system and highlight mast cell activators as a new class of vaccine adjuvants. Herman Staats and colleagues (Durham, NC) studied the adjuvant properties of the mast cell activator compound 48/80 which, when nasally delivered with various protein Ag, induced immune responses comparable to those induced by the adjuvant cholera toxin, the gold Dolichyl-phosphate-mannose-protein mannosyltransferase standard mucosal adjuvant 34, 35. Dr. Staats found that compound 48/80 was as effective as cholera toxin for the induction of serum IgG and mucosal IgA against vaccine Ag. As a nasal vaccine adjuvant, compound 48/80 enhanced anthrax lethal toxin neutralizing antibody titers and protection against a lethal vaccinia virus challenge in the absence of adverse effects such as induction of Ag-specific IgE. When delivered by the intradermal route, compound 48/80 induced a balanced Th1/Th2 response as well as heightened IgG, but not IgE, antibody responses. These results suggest that mast cell activators represent a new class of adjuvants that may be safely administered with intradermal or intranasal vaccines.

No relationship between TNF-α polymorphism and SBI susceptibility was found in this study. Alzheimer’s disease (AD) is one of the most common types of chronic neurodegenerative diseases. Vascular dementia, AD and stroke are all associated with inflammation, but they have different initiating factors. Polymorphism in the TNF and apolipo protein E (APOE) was reported to increase AD risk. Laws Simon et al. [128] conducted a case–control

study and investigated −850C>T, rs1800629 in TNF and the APOE polymorphism in controls and patients with sporadic AD. The frequency of (−850C/T) genotypes and T allele was significantly different in AD individuals, while the (rs1800629) SNP was not associated with AD. Venetoclax nmr T allele of (−850) polymorphism significantly modified risk associated with possession of the APOE e4 allele only,

and (−850) T allele was found to be associated with lower levels of CSF Aβ42. In a Southern China population, patients with sporadic Alzheimer’s disease (SAD) have a significantly increased frequency of rs1800629 A-allele as compared with controls. The carriers of A-allele have a significantly increased risk of SAD. Level of TNF-α in serum of SAD group was much higher than that in control group, and the elevated serum MLN0128 chemical structure TNF-alpha level was closely associated with the risk of SAD detected by Yang et al. [129]. Seventeen studies that investigated the association between five TNF-α polymorphism (−850, rs1800629, rs1800630, rs361525 and rs1799964) and AD were retrieved and analysed [130]. The presence of T allele significantly increased the risk of AD associated with carriage of the apolipoprotein E epsilon 4 allele in Caucasian Australians and Northern Europeans. A significant association of (−850) polymorphism with AD risk and non-significant difference in genotype distribution of (rs1800629)

polymorphism in AD was found. Farnesyltransferase For the (rs361525 and rs1799964) polymorphism, Di Bona et al. [130] did not find an association with AD. Only four studies investigated rs361525 variant, and the results were not significant. Current findings suggested an association between (−850C>T) polymorphism and the risk of developing AD. No positive associations between TNF-alpha promoter haplotypes and AD disease in Italian population have been reported by Tedde et al. [131]. Tumour necrosis factor plays an important role in glutamatergic neural transmission [132] and serve essential functions in neural plasticity [133] and cognitive processes like learning and memory [134, 135]. SNPs in TNF have profound impact on this disease. A-allele of rs1800629 fastens cognitive processing speed in a visual task, compared with G-allele carriers [136]. Mental rotation describes the cognitive process of imagining an object turning around. Mental rotation is usually examined using objects (e.g. letters) that are rotated by certain degrees clockwise or counter-clockwise from the vertical upright.

Submicroscopic infections that are highly prevalent in all malaria endemic settings [31] appeared to provide sufficiently high levels of antigen exposure to maintain

Selleck BAY 57-1293 antibody titres. Our findings confirm observations in Kenyan children where antibody boosting was observed in the absence of patent malaria infections and provide evidence in support of their hypothesis that this could be explained by submicroscopic infections [32]. Our data also offer support for the hypothesis that circulating antimalarial antibodies in children derive mainly from short-lived plasma cells [33] but that long-lived plasma cells may be the major source of antibodies in older individuals [34]. Finally, the very rapid decline – in all age groups – in titres of antibodies to mosquito salivary gland antigens indicates that these antigens fail to induce long-lived plasma cells, suggesting that the antibodies may emanate from ‘innate’ or ‘natural’ B1 cells or that the antigens activate B cells in a T-cell

independent manner (35). We are grateful to the Apac district’s inhabitants for their participation to the study; we also thank BMS-777607 order Sam Edweo and Dorcus Akello for their contribution during the field work. This study was supported by the FIGHTMAL project, receiving funding from the European Community’s Seventh Framework Programme [FP7/2007-2013] under grant agreement PIAP-GA-2008-218164. “
“In certain infection sites or tumor tissues, the disruption of homeostasis can give rise to a hypoxic microenvironment, which, in turn, can alter

the function of different immune cell types and favor the progression of the disease. Natural killer (NK) cells are directly involved in the elimination of virus-infected or transformed cells, however it is unknown whether their function is affected by hypoxia or not. In this study, we show that NK cells adapt to a hypoxic buy ZD1839 environment by upregulating the hypoxia-inducible factor 1α. However, NK cells lose their ability to upregulate the surface expression of the major activating NK-cell receptors (NKp46, NKp30, NKp44, and NKG2D) in response to IL-2 (or other activating cytokines, including IL-15, IL-12, and IL-21). These altered phenotypic features correlate with reduced responses to triggering signals resulting in impaired capability of killing infected or tumor target cells. Remarkably, hypoxia does not significantly alter the surface density and the triggering function of the Fc-γ receptor CD16, thus allowing NK cells to maintain their capability of killing target cells via antibody-dependent cellular cytotoxicity. This finding offers an important clue for exploitation of NK cell in antibody-based immunotherapy of cancer. As a component of innate immunity, natural killer (NK) cells play an important role in the control of virus infections and in cancer immune surveillance [1-5].

3D). Finally, to confirm that LTi-like cell survival in vitro did not require IL-7, LTi prepared

from Rag−/−γc−/− mice (therefore unresponsive to IL-7) were cultured with CD45−podoplanin+ SSCL and in this assay survival was not affected (Fig. 3C). To examine whether adult LTi-like cells were able to survive without IL-7 or γc cytokine survival factors within CT99021 the splenic T zone in vivo, tissue sections of spleen from IL-7−/− and Rag−/−γc−/− mice were stained for LTi-like cells and analyzed by immunofluorescent confocal microscopy. In both types of mice, LTi-like cells were distributed within the splenic T zones and these cells were in close proximity with VCAM-1+ stroma of T-cell areas suggesting interactions between LTi-like cells and surrounding T-zone stroma (Fig. 3E). Together, these data provide evidence that splenic adult LTi-like cells are able to survive in vivo in an IL-7-independent manner. In the embryo, IL-7 is important in controlling numbers of LTi. In transgenic check details mice expressing high levels of IL-7, LTi numbers are greatly increased, resulting in increased numbers of Peyer’s patches and ectopic LN 20. Current data indicate that IL-7 improves embryonic LTi survival. Our previous studies have demonstrated that LTi persist in the adult spleen of IL-7−/− and γc−/− mice 18. In this study we identified IL-7Rα+ and IL-7Rα−

LTi-like cells in adult spleen. We found that CD45−podoplanin+ SSCL promote the survival of adult LTi-like cells in an IL-7 independent manner, and that LTi-like cells isolated from Rag−/−γc−/− Aurora Kinase mice survived well in culture with CD45−podoplanin+ SSCL. Finally, splenic LTi-like cells exist in IL-7−/− and γc−/− mice in situ and these cells remain in the white pulp areas where they interact with VCAM-1+ T-zone stroma which express podoplanin. Taken together these data suggest that stromal-derived factors control the survival

of LTi in the adult. While IL-7 plays a key role in vivo, there appears to be redundancy in the signals supporting LTi-like cell survival in the adult spleen. All experiments were performed in accordance with UK laws and with the approval of the University of Birmingham ethics committee. C57Bl6, RAG2−/−, RAG2−/−/commonγchain−/− (Rag−/−γc−/−) and CD3ε-transgenic (CD3εtg) mice were all bred and maintained in the Biomedical Services Unit at the University of Birmingham. CD3εtg mice have a profound block in the early T-cell development at the CD4−CD8−CD25−CD44+ stage in the thymus and display a complete loss of T-lymphocytes 21. Mouse spleens were excised and digested in RPMI 1640 medium with 10% FCS plus 1 mg/mL collagenase/dispase (Roche) and 20 μg/mL DNase 1 (Sigma) at 37°C for 20 min. To aid digestion, tissues were agitated to disperse aggregates.

2c). A higher magnification in these areas revealed biofilm clusters consisting of live and dead cocci surrounded by EPS containing eDNA (Fig. 2d). Although it has long been recognized that monofilament sutures may generally harbor fewer microorganisms than multifilament sutures (e.g. Osterberg & Blomstedt, 1979), these

striking images show that the knotted area itself, unavoidable with any suture configuration, can provide an adequate microenvironment in which biofilm may accumulate. In light of the above findings, the patient’s clinical history is thrown into sharper relief and is consistent with the biofilm paradigm, fulfilling all of Parsek and

Singh’s suggested criteria for the clinical diagnosis of a biofilm infectious process (Parsek & Singh, 2003). These include: ‘(a) The infecting bacteria were adherent to some substratum Ibrutinib supplier or are surface associated’– clearly, in this case, bacteria were adherent to the xenograft and to the sutures, as demonstrated by CM. ‘(b) Direct examination of infected tissue shows bacteria living in cell clusters, or microcolonies, encased in an extracellular matrix’– again, our confocal results show just this. ‘(c) The infection is generally confined to a particular location. SCH772984 Although dissemination may occur, it is a secondary phenomenon’– the present case is a particularly good example of this. On the patient’s left side, despite months of pain (now understood to be the result of an infectious process), no systemic spread occurred; nor was the infection visible externally. We suspect the patient likely had a similar biofilm-elicited process on the right side that did progress to development of a frank draining sinus, but even this remained a localized process, with no cellulitic or systemic spread over months. ‘(d) The infection is difficult or impossible to eradicate with antibiotics

despite the fact that the responsible organisms are susceptible to killing in the planktonic state’– this characteristic was never tested in this patient. Because we suspected a biofilm FER etiology to the patient’s infections, we relied on surgical exploration rather than antibiosis as the mainstay of intervention. Antibiotics were only administered adjuvantly, after the substrata hosting the biofilms were surgically removed. This case also conforms to other typical features of biofilm infections. Despite numerous bacteria present and visible on explanted xenograft tissues, laboratory culture was positive in only one instance, consistent with the difficulty in recovering biofilm organisms using standard microbiological cultural techniques.