Mapping the colorectal tumor microbiota.

The gut microbiome in patients with colorectal cancer (CRC) is different than that of healthy controls. Previous studies have profiled the CRC tumor microbiome using a single biopsy. However, since the morphology and cellular subtype vary significantly within an individual tumor, the possibility of sampling error arises for the microbiome within an individual tumor. To test this hypothesis, seven biopsies were taken from representative areas on and off the tumor in five patients with CRC. The microbiome composition was strikingly similar across all samples from an individual. The variation in microbiome alpha-diversity was significantly greater between individuals' samples then within individuals. This is the first study, to our knowledge, that shows that the microbiome of an individual tumor is spatially homogeneous. Our finding strengthens the assumption that a single biopsy is representative of the entire tumor, and that microbiota changes are not limited to a specific area of the neoplasm.

CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation.

OBJECTIVE: To investigate the efficacy of CRISPR-Cas9 mediated editing in human chondrocytes, and to develop a genome editing approach relevant to cell-based repair. METHODS: Transfection of human articular chondrocytes (both healthy and osteoarthritic) with ribonucleoprotein complexes (RNP) containing Cas9 and a crisprRNA targeting exon2 of MMP13 was performed to assess editing efficiency and effects on MMP13 protein levels and enzymatic activity. Using spheroid cultures, protein levels of a major target of MMP13, type II collagen, were assessed by western blot and immunofluorescence. RESULTS: With an editing efficiency of 63-74%, secreted MMP13 protein levels and activity were significantly reduced (percentage decrease 34.14% without and 67.97% with IL-1ß based on median values of MMP13 enzymatic activity, N = 7) comparing non-edited with edited cell populations using an exon-targeting gRNA resulting in premature stop codons through non-homologous end joining (NHEJ). Accumulation of cartilage matrix protein type II collagen was enhanced in edited cells in spheroid culture, compared to non-edited controls. CONCLUSION: CRISPR-Cas9 mediated genome editing can be used to efficiently and reproducibly establish populations of human chondrocytes with stably reduced expression of key genes of interest without the need for clonal selection. Such an editing approach has the potential to greatly enhance current cell-based therapies for cartilage repair.

Major cost savings associated with biologic dose reduction in patients with inflammatory arthritis.

The purpose of this study was to explore whether patients with Inflammatory Arthritis (IA) (Rheumatoid Arthritis (RA), Psoriatic Arthritis (PsA) or Ankylosing Spondylitis (AS)) would remain in remission following a reduction in biologic dosing frequency and to calculate the cost savings associated with dose reduction. This prospective non-blinded non-randomised study commenced in 2010. Patients with Inflammatory Arthritis being treated with a biologic agent were screened for disease activity. A cohort of those in remission according to standardized disease activity indices (DAS28 < 2.6, BASDAI < 4) was offered a reduction in dosing frequency of two commonly used biologic therapies (etanercept 50 mg once per fortnight instead of weekly, adalimumab 40 mg once per month instead of fortnightly). Patients were assessed for disease activity at 3, 6, 12, 18 and 24 months following reduction in dosing frequency. Cost saving was calculated. 79 patients with inflammatory arthritis in remission were recruited. 57% had rheumatoid arthritis (n = 45), 13% psoriatic arthritis (n = 10) and 30% ankylosing spondylitis (n = 24). 57% (n = 45) were taking etanercept and 43% (n = 34) adalimumab. The percentage of patients in remission at 24 months was 56% (n = 44). This resulted in an actual saving to the state of approximately 600,000 euro over two years. This study demonstrates the reduction in biologic dosing frequency is feasible in Inflammatory Arthritis. There was a considerable cost saving at two years. The potential for major cost savings in biologic usage should be pursued further.

Lithium in patients with amyotrophic lateral sclerosis (LiCALS): a phase 3 multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Lithium has neuroprotective effects in cell and animal models of amyotrophic lateral sclerosis (ALS), and a small pilot study in patients with ALS showed a significant effect of lithium on survival. We aimed to assess whether lithium improves survival in patients with ALS. METHODS: The lithium carbonate in amyotrophic lateral sclerosis (LiCALS) trial is a randomised, double-blind, placebo-controlled trial of oral lithium taken daily for 18 months in patients with ALS. Patients aged at least 18 years who had ALS according to the revised El Escorial criteria, had disease duration between 6 and 36 months, and were taking riluzole were recruited from ten centres in the UK. Patients were randomly assigned (1:1) to receive either lithium or matched placebo tablets. Randomisation was via an online system done at the level of the individual by block randomisation with randomly varying block sizes, stratified by study centre and site of disease onset (limb or bulbar). All patients and assessing study personnel were masked to treatment assignment. The primary endpoint was the rate of survival at 18 months and was analysed by intention to treat. This study is registered with Eudract, number 2008-006891-31. FINDINGS: Between May 26, 2009, and Nov 10, 2011, 243 patients were screened, 214 of whom were randomly assigned to receive lithium (107 patients) or placebo (107 patients). Two patients discontinued treatment and one died before the target therapeutic lithium concentration could be achieved. 63 (59%) of 107 patients in the placebo group and 54 (50%) of 107 patients in the lithium group were alive at 18 months. The survival functions did not differ significantly between groups (Mantel-Cox log-rank χ(2) on 1 df=1·64; p=0·20). After adjusting for study centre and site of onset using logistic regression, the relative odds of survival at 18 months (lithium vs placebo) was 0·71 (95% CI 0·40-1·24). 56 patients in the placebo group and 61 in the lithium group had at least one serious adverse event. INTERPRETATION: We found no evidence of benefit of lithium on survival in patients with ALS, but nor were there safety concerns, which had been identified in previous studies with less conventional designs. This finding emphasises the importance of pursuing adequately powered trials with clear endpoints when testing new treatments. FUNDING: The Motor Neurone Disease Association of Great Britain and Northern Ireland.

Benefits of pre-referral preparation for rheumatology clinics.

Appropriate allocation of rheumatology clinic appointments depends on the information contained in referral letters. Such letters were analysed for the presence of pertinent information and a scoring system was devised to assess the quality of enclosed data. In a smaller cohort, relevant basic tests were carried out prior to the appointment. 122 referral letters were received over a 1 month period. Symptom duration was documented in (39) 32%, while (64) 52.5% listed medications. Only (23) 17.2% indicated the urgency of the problem. Approximately (31) 25% of referrers performed relevant routine investigations. Mean score out of 10 was 5.1 (range 1.5-9). Of the 40 (33%) patients with pre-appointment investigations, the clinic attendance rate and subsequent discharge rate were significantly higher than those without these tests. This study shows that comprehensive referral letters and basic investigations significantly help to prioritize appointments and facilitate earlier diagnosis and treatment for patients with rheumatic disease.

Immunohistochemical and biochemical evidence of ameloblastic origin of amyloid-producing odontogenic tumors in cats.

Amyloid-producing odontogenic tumors (APOT) are rare, and in cats, the histogenesis of the amyloid remains undetermined. In the present study, APOTs in 3 cats were characterized by immunohistochemistry, and the amyloid components analyzed using tandem mass spectrometry. Antiameloblastin antibodies labeled both neoplastic epithelial cells and amyloid in all cases. Neoplastic epithelial cells had strong, diffuse immunoreactivity to antibodies against cytokeratin AE1/AE3, cytokeratin 14, and cytokeratin 19 in all cases and focal immunoreactivity to nerve growth factor receptor antibodies in 2 of 3 cases. Amyloid and some tumor stromal cells were weakly positive for laminin. Calretinin, amelogenin, S100, and glial fibrillary acidic protein antibodies did not label neoplastic epithelial cells or amyloid. Extracted amyloid peptide sequences were compared to the porcine database because the cat genome is not yet complete. Based on this comparison, 1 identical ameloblastin peptide was detected in each tumor. These results suggest that feline APOTs and the amyloid they produce are of ameloblastic lineage.

Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains.

AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescence microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

The identification of differentially expressed microRNA in osteoarthritic tissue that modulate the production of TNF-alpha and MMP13.

OBJECTIVE: To identify differentially expressed microRNAs (miRNAs) in human osteoarthritic (OA) cartilage and bone tissue and to determine their relevance to chondrocyte function. METHODS: Cartilage and bone was obtained from OA patients who underwent total knee joint replacement surgery or from post-mortem patients with no previous history of OA. MiRNA expression was quantified by real-time PCR (RT-PCR). Functional pathway analysis of miRNA was performed using Ingenuity Pathway Analysis. Primary chondrocytes were isolated by collagenase digestion and transfected with miRNA mimics and miRNA inhibitors using cationic lipid. Tumour Necrosis Factor-alpha (TNF-alpha) and Matrix metalloprotease 13 (MMP13) protein levels were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS: In total we identified 17 miRNA that showed greater than 4-fold differential expression between OA and normal cartilage, and 30 miRNA that showed greater than 4-fold differential expression in OA bone. Functional pathway analysis of the predicted gene targets for miR-9, miR-98, which were upregulated in both OA bone and cartilage tissue, and miR-146, which was downregulated in OA cartilage, suggested that these miRNA mediate inflammatory functions and pathways. Over-expression of miR-9, miR-98 or miR-146 in isolated human chondrocytes reduced interleukin-1 beta (IL-1 beta) induced TNF-alpha production. Furthermore, inhibition and over-expression of miR-9 modulated MMP13 secretion. CONCLUSIONS: We have identified a number of differentially expressed miRNAs in late-stage human OA cartilage and bone. Functional analysis of miR-9, miR-98 and miR-146 in primary chondrocytes suggests a role in mediating the IL-1 beta induced production of TNF-alpha. MiR-9, upregulated in OA tissue, was found to inhibit secretion of the collagen type II-targeting metalloproteinase MMP13 in isolated human chondrocytes.

Topographical variation in glycosaminoglycan content in human articular cartilage.

The weight-bearing status of articular cartilage has been shown to affect its biochemical composition. We have investigated the topographical variation of sulphated glycosaminoglycan (GAG) relative to the DNA content of the chondrocyte in human distal femoral articular cartilage. Paired specimens of distal femoral articular cartilage, from weight-bearing and non-weight-bearing regions, were obtained from 13 patients undergoing above-knee amputation. After papain enzyme digestion, spectrophotometric GAG and fluorometric DNA assays assessed the biochemical composition of the samples. The results were analysed using a paired t-test. Although there were no significant differences in cell density between the regions, the weight-bearing areas showed a significantly higher concentration of GAG relative to DNA when compared with non-weight-bearing areas (p = 0.02). We conclude that chondrocytes are sensitive to their mechanical environment, and that local loading conditions influence the metabolism of the cells and hence the biochemical structure of the tissue.

Systemic AA amyloidosis in the common marmoset.

The common marmoset (Callithrix jacchus) is a small New World primate native to Brazil that has been used extensively in biomedical research. A retrospective analysis of archived hematoxylin and eosin-stained tissue sections and clinical records was conducted at the New England Primate Research Center on 86 marmosets more than 1 year of age that were euthanized during the past decade because of morbidity and failure to thrive. Approximately 17% (15 of 86) were found to have amyloid deposits in one or more organs, including the liver, adrenal glands, kidneys, and intestine. This material was shown by amino acid sequence analysis to be composed of serum amyloid A (SAA)-related protein. This type of amyloidosis, designated AA or "secondary," is associated typically with an inflammatory process that induces elevated levels of the SAA amyloidogenic precursor molecule. Notably, there were no significant pathologic differences or other distinguishing features in animals with amyloid versus those without; furthermore, on the basis of the limited number of serum specimens available for analysis, the SAA concentrations in the two groups were comparable, thus suggesting the possible inheritable nature of the disorder. In this respect, the common marmoset provides a unique experimental model for study of the pathogenesis and treatment of AA and other forms of systemic amyloidosis.

Pathology of AA amyloidosis in domestic sheep and goats.

We describe the main pathologic changes in small ruminants affected by AA amyloidosis, together with the partial sequence of the protein involved. Twenty-one sheep and one goat were selected for presenting macroscopic kidney lesions compatible with systemic amyloidosis. Available tissue samples were studied by histologic, immunopathologic, and ultrastructural means. Renal lesions were characterized grossly by pale cortical surfaces with scattered, miliary, whitish-yellow foci and on cut cortical surfaces by straight, whitish-yellow striations. Gangrenous pneumonia was observed in 16 out of 21 affected sheep (76.2%), although other chronic inflammations were also observed. Amyloid was detected in all grossly affected kidneys using Congo red staining, lesions being most remarkable in glomeruli, affecting 95.5% of animals studied. Congophilic deposits were also observed in intertubular interstitium (68.2%) and medulla (57.1%). All amyloid-affected animals presented proximal convoluted tubule lesions, mostly characterized by an increase in diameter and by hyaline granular degeneration that were responsible for the macroscopic appearance of the kidney. Histologically, amyloid was also seen in blood vessels, spleen, liver, lymph nodes, gastrointestinal tract, and adrenal glands. All amyloid deposits demonstrated greenish-yellow birefringence with polarized light, and the antisera prepared against goat amyloid extracts specifically reacted with birefringent congophilic deposits of both sheep and goats. Ultrastructurally, these deposits were formed by masses of straight, nonbranching fibrils located predominantly in the basement membranes of glomerular capillaries and in the mesangium. Partial sequence of the protein in sheep and goats indicated a high degree of homology with the previously reported sequence of sheep Serum Amyloid A.

Effect of oxygen tension and alginate encapsulation on restoration of the differentiated phenotype of passaged chondrocytes.

The implantation of laboratory-grown tissue offers a valuable alternative approach to the treatment of cartilage defects. Procuring sufficient cell numbers for such tissue-engineered cartilage is a major problem since amplification of chondrocytes in culture typically leads to loss of normal cell phenotype yielding cartilage of inferior quality. In an effort to overcome this problem, we endeavored to regain the differentiated phenotype of chondrocytes after extensive proliferation in monolayer culture by modulating cell morphology and oxygen tension towards the in vivo state. Passaged cells were encapsulated in alginate hydrogel in an effort to regain the more rounded shape characteristic of differentiated chondrocytes. These cultures were exposed to reduced (5%-i.e., physiological), or control (20%) oxygen tensions. Both alginate encapsulation and reduced oxygen tension significantly upregulated collagen II and aggrecan core protein expression (differentiation markers). In fact, after 4 weeks in alginate at 5% oxygen, differentiated gene expression was comparable to primary chondrocytes. Collagen I expression (dedifferentiation marker) decreased dramatically after alginate entrapment, while reduced oxygen tension had no effect. It is concluded that alginate encapsulation and reduced oxygen tension help restore key differentiated phenotypic markers of passaged chondrocytes. These findings have important implications for cartilage tissue engineering, since they enable the increase in differentiated cell numbers needed for the in vitro development of functional cartilaginous tissue suitable for implantation.

Effect of oxygen tension on chondrocyte extracellular matrix accumulation.

Since cartilage is mainly an avascular tissue, chondrocytes exist in a low-level oxygen environment in vivo. In the present study, we investigated the effect of oxygen tension (20%, 5% and 1% gas phase oxygen concentrations) over a 20-day period on the extracellular matrix accumulation of bovine articular chondrocytes in confluent surface cultures. Matrix accumulation was assessed by the amount of glycosaminoglycan and collagen deposited in the matrix. From initially confluent monolayers, the chondrocytes became distributed throughout a thick layer of extracellular matrix, thus forming a multicell-layer of tissue. Cells maintained their normal rounded shape, indicative of the differentiated phenotype, throughout the 20-day culture period. On a per culture and a per cell basis, the amount of collagen and glycosaminoglycan accumulation in the matrix was lower at the reduced oxygen tensions. Specifically, in 1% oxygen, matrix GAG content reached a steady-state level, with no net increase in GAG levels after two weeks, whereas in 20% oxygen, matrix GAG increased with time. It is concluded that oxygen has a significant effect on the amount of macromolecules accumulated in the extracellular matrix. The implications of these findings in growing cartilage constructs in vitro are discussed.

A placebo-controlled trial to evaluate immunomodulatory effects of paricalcitol.

Calcitriol has shown a benefit in various small uncontrolled studies of ex vivo immune function. We hypothesized that paricalcitol, a new vitamin D derivative, will have a positive effect on the immune system with minimal adverse effects on calcium homeostasis. Thirty-one hemodialysis patients not administered vitamin D because of low intact parathyroid hormone (PTH) levels were randomized to placebo or 4 microg of paricalcitol intravenously with the hemodialysis session three times weekly for 12 weeks. Effects on in vivo and ex vivo assessments of immune function were evaluated. All patients achieved the target dose of paricalcitol. Twenty patients were anergic at the start of the study; 4 of 11 patients in the paricalcitol group and 0 of 9 patients in the placebo group converted to reactive (P = 0.09). The in vivo response to standard hepatitis B booster vaccine and in vitro proliferation and release of interleukin-2 (IL-2), IL-6, tumor necrosis factor-alpha, and interferon-gamma from stimulated lymphocytes were not different between the groups. In contrast to clinical immune effects, paricalcitol increased serum calcium levels and decreased PTH and bone alkaline phosphatase levels (all P < 0.05). However, hypercalcemia was infrequent. In vitro experiments showed that paricalcitol led to greater dose-dependent thymidine uptake than calcitriol in lymphocytes isolated from either dialysis patients or control subjects. Paricalcitol has a tendency toward improving delayed hypersensitivity reactions, but did not have other proimmune effects. However, as expected, paricalcitol had significant effects on calcium homeostasis compared with placebo. Thus, patients with low PTH levels are unlikely to experience the proimmune effects of vitamin D therapy without more profound and potentially adverse oversuppression of PTH.

Variation in administrators' and clinicians' attitudes toward critical elements of an infection control program and the role of the infection control practitioner in New South Wales, Australia.

BACKGROUND: Debate remains over the core activities of infection control (IC) programs. Differences in stakeholder opinions must be considered if consensus panel guidelines and recommendations are to be broadly applied. This article describes a survey of administrators and clinicians employed in hospitals in New South Wales, Australia. Respondents self-reported their levels of agreement with affirmative statements regarding the role of the infection control practitioner (ICP) and the essential requirements and infrastructure of IC programs. METHOD: The study population included administrators and clinicians in each public, private, and freestanding day hospital in New South Wales. Respondents reported the intensity of their agreement with 16 affirmative statements relating to IC program infrastructure and resources and the ICP's role and responsibilities. RESULTS: The overall response rate was 62.1% (587/945). Clinicians (349/587) and administrators (238/587) accounted for 59.5% and 40.5% of the response rate, respectively. Overall, administrators and clinicians reported greatest levels of agreement for those elements not requiring additional resources. CONCLUSION: The extent of divergence between administrators and clinicians is not so great that it can not be resolved. Our findings demonstrate the degree of administrator support that clinicians can expect for each element. We advocate better communication between clinicians and administrators in conjunction with objective strategic planning. Our findings provide a guide for ICPs to either establish or negotiate the core components of their IC program.

Chemical typing of amyloid protein contained in formalin-fixed paraffin-embedded biopsy specimens.

The human amyloidoses represent a heterogeneous group of disorders characterized by the deposition of fibrillar protein in vital organs. Given the fact that at least 20 different molecules can form fibrils, the unambiguous identification of the type of amyloid deposited is critical to the correct diagnosis and treatment of patients with these disorders. Heretofore, this information has been inferred from particular clinical features of the disease, ancillary laboratory tests, and results of immunohistochemical analyses. However, to establish unequivocally the kind of protein that is deposited as amyloid, it is necessary to determine its chemical composition through amino acid sequencing or mass spectroscopy of material extracted from fibrillar deposits. We have developed a micromethod whereby such studies can be performed readily using sections of formalin-fixed, paraffin-embedded biopsy specimens. The ability to identify precisely the nature of the tissue deposits has diagnostic, therapeutic, and prognostic implications for patients with amyloid-associated disorders.

Micro-method to isolate and purify amyloid proteins for chemical characterization.

The amyloidoses represent a heterogeneous group of disorders characterized by the pathologic deposition as fibrils of at least 20 different precursor molecules. To establish definitively the specific type of amyloid protein contained in fibrillar deposits, such material must be extracted, purified, and subjected to amino acid sequence analysis. Heretofore, the chemical identification of amyloid components has required gram quantities of tissue. Given the often-limited amounts of sample available, e.g., that derived from diagnostic needle biopsies, we have developed a micro-method to isolate and purify amyloid from minute tissue specimens. The procedure involves micro-extraction of the amyloid with subsequent purification by SDS-PAGE, electroblotting onto PVDF membranes, excision and elution of amyloid protein-related bands, and reversed phase HPLC. Chemical and immunologic studies of isolated amyloid components have demonstrated the purity achieved with this technique and have provided information on the molecular mass, heterogeneity, and immunoreactivity of the amyloid. Further, using this methodology, it has been possible to obtain sufficient material for amino acid sequencing and thus to establish unequivocally the chemical and molecular composition of the fibrillar deposits. Our microtechnique has clinical import and also is applicable to analyses of the amyloid found in experimental small animal models of these disorders.