Noroviruses as a Cause of Diarrhea in Immunocompromised Pediatric Hematopoietic Stem Cell and Solid Organ Transplant Recipients.

Case reports describe significant norovirus gastroenteritis morbidity in immunocompromised patients. We evaluated norovirus pathogenesis in prospectively enrolled solid organ (SOT) and hematopoietic stem cell transplant (HSCT) patients with diarrhea who presented to Texas Children's Hospital and submitted stool for enteric testing. Noroviruses were detected by real-time reverse transcription polymerase chain reaction. Clinical outcomes of norovirus diarrhea and non-norovirus diarrhea patients, matched by transplanted organ type, were compared. Norovirus infection was identified in 25 (22%) of 116 patients, more frequently than other enteropathogens. Fifty percent of norovirus patients experienced diarrhea lasting ≥14 days, with median duration of 12.5 days (range 1-324 days); 29% developed diarrhea recurrence. Fifty-five percent of norovirus patients were hospitalized for diarrhea, with 27% requiring intensive care unit (ICU) admission. One HSCT recipient developed pneumatosis intestinalis. Three HSCT patients expired ≤6 months of norovirus diarrhea onset. Compared to non-norovirus diarrhea patients, norovirus patients experienced significantly more frequent ICU admission (27% vs. 0%, p = 0.02), greater serum creatinine rise (median 0.3 vs. 0.2 mg/dL, p = 0.01), and more weight loss (median 1.6 vs. 0.6 kg, p < 0.01). Noroviruses are an important cause of diarrhea in pediatric transplant patients and are associated with significant clinical complications.

Clinical predictors of disease severity during the 2009-2010 A(HIN1) influenza virus pandemic in a paediatric population.

A novel influenza virus emerged in the United States in spring 2009, rapidly becoming a global pandemic. Children were disproportionally affected by the novel influenza A(H1N1) pandemic virus [A(H1N1)pdm]. This retrospective electronic medical record review study aimed to identify clinical predictors of disease severity of influenza A(HIN1)pdm infection in paediatric patients. Disease severity was defined on an increasing three-level scale from non-hospitalized, hospitalized, and admitted to the intensive care unit (ICU). From April 2009 to June 2010, 696 children presented to Texas Children's Hospital's emergency department, 38% were hospitalized, and 17% were admitted to the ICU. Presenting symptoms associated with severe influenza were dyspnoea [odds ratio (OR) 5·82], tachycardia (OR 2·61) and fatigue (OR 1·96). Pre-existing health conditions associated with disease severity included seizure disorder (OR 4·71), obesity (OR 3·28), lung disease (OR 2·84), premature birth (OR 2·53), haematological disease (OR 2·22), and developmental delay (OR 2·20). According to model fitness tests, presenting symptoms were more likely to predict severe influenza than underlying medical conditions. However, both are important risk factors. Recognition of clinical characteristics associated with severe disease can be used for triaging case management of children during future influenza outbreaks.

Challenges of Francisella classification exemplified by an atypical clinical isolate.

The accumulation of sequenced Francisella strains has made it increasingly apparent that the 16S rRNA gene alone is not enough to stratify the Francisella genus into precise and clinically useful classifications. Continued whole-genome sequencing of isolates will provide a larger base of knowledge for targeted approaches with broad applicability. Additionally, examination of genomic information on a case-by-case basis will help resolve outstanding questions regarding strain stratification. We report the complete genome sequence of a clinical isolate, designated here as F. novicida-like strain TCH2015, acquired from the lymph node of a 6-year-old male. Two features were atypical for F. novicida: exhibition of functional oxidase activity and additional gene content, including proposed virulence determinants. These differences, which could potentially impact virulence and clinical diagnosis, emphasize the need for more comprehensive methods to profile Francisella isolates. This study highlights the value of whole-genome sequencing, which will lead to a more robust database of environmental and clinical genomes and inform strategies to improve detection and classification of Francisella strains.

Increased expression of inducible co-stimulator on CD4+ T-cells in the peripheral blood and synovial fluid of patients with failed hip arthroplasties.

OBJECTIVES: T-cells are considered to play an important role in the inflammatory response causing arthroplasty failure. The study objectives were to investigate the composition and distribution of CD4+ T-cell phenotypes in the peripheral blood (PB) and synovial fluid (SF) of patients undergoing revision surgery for failed metal-on-metal (MoM) and metal-on-polyethylene (MoP) hip arthroplasties, and in patients awaiting total hip arthroplasty. METHODS: In this prospective case-control study, PB and SF were obtained from 22 patients (23 hips) undergoing revision of MoM (n = 14) and MoP (n = 9) hip arthroplasties, with eight controls provided from primary hip osteoarthritis cases awaiting arthroplasty. Lymphocyte subtypes in samples were analysed using flow cytometry. RESULTS: The percentages of CD4+ T-cell subtypes in PB were not different between groups. The CD4+ T-cells in the SF of MoM hips showed a completely different distribution of phenotypes compared with that found in the PB in the same patients, including significantly decreased CD4+ T-central memory cells (p < 0.05) and increased T-effector memory cells (p < 0.0001) in the SF. Inducible co-stimulator (ICOS) was the only co-stimulatory molecule with different expression on CD4+ CD28+ cells between groups. In PB, ICOS expression was increased in MoM (p < 0.001) and MoP (p < 0.05) cases compared with the controls. In SF, ICOS expression was increased in MoM hips compared with MoP hips (p < 0.05). CONCLUSIONS: Increased expression of ICOS on CD4+ T-cells in PB and SF of patients with failed arthroplasties suggests that these cells are activated and involved in generating immune responses. Variations in ICOS expression between MoM and MoP hips may indicate different modes of arthroplasty failure.Cite this article: Professor P. A. Revell. Increased expression of inducible co-stimulator on CD4+ T-cells in the peripheral blood and synovial fluid of patients with failed hip arthroplasties. Bone Joint Res 2016;5:52-60. doi: 10.1302/2046-3758.52.2000574.

The use of foetal calf serum as a blocking agent in double immunoenzyme-cytochemistry labelling with mouse monoclonal antibodies.

Double immunoenzyme labelling has progressed in the past few years to double labelling using monoclonal antibodies (MAB) raised in the same species. It presents the problem of cross-reaction between the primary antibodies and the enzyme-labelled secondary antibodies as well as that of excessively high background staining. These difficulties have been overcome in part by using normal mouse serum as a blocking agent. However, differentiation of the resulting colour reactions, and therefore interpretation of results, is difficult, especially when compounded by the presence of an intermediate colour due to the combination of two reactions. Because of this problem, normal mouse serum (NMS), BALB/c mouse serum and foetal calf serum (FCS) were tested for their effectiveness as blocking agents in a double-labelling procedure using two mouse anti-human MABs.

Chronic acidosis with metabolic bone disease. Effect of alkali on bone morphology and vitamin D metabolism.

Chronic metabolic acidosis and osteomalacia developed in two patients following urinary diversion. Good clinical, biochemical, and histologic responses were seen following treatment with alkali alone (vitamin D was not given), despite the presence of markedly impaired glomerular filtration in one of the patients. Plasma 25-hydroxyvitamin D and 1 alpha, 25-dihydroxyvitamin D concentrations were normal before and during treatment in one of the patients and in the other were low before and normal during treatment. The results show that successful treatment of the osteomalacia of chronic acidosis is not necessarily accompanied by changes in the plasma levels of vitamin D metabolites and that even when marked glomerular dysfunction coexists with acidosis and osteomalacia, treatment with alkali may be more appropriate than the administration of vitamin D analogues.

Substance P-, calcitonin gene-related peptide- and C-flanking peptide of neuropeptide Y-immunoreactive fibres are present in normal synovium but depleted in patients with rheumatoid arthritis.

By means of antisera to cytoplasmic components of nerve fibres and neuropeptides which are known to be present in sensory or sympathetic nerves we have examined the distribution of both total and different types of nerve fibres in normal and inflamed human synovial tissue. Samples of synovia were obtained at surgery from five normal and five rheumatoid patients (age range 10-77 years). In order to map the overall neural innervation of the synovium, antiserum to the general neuronal marker protein gene product 9.5 was employed. Substance P and calcitonin gene-related peptide antisera were employed to identify sensory fibres and antisera to the C-flanking peptide of neuropeptide Y to distinguish sympathetic nerves. In normal synovium protein gene product 9.5-immunoreactive fibres were numerous, in particular, the vasculature was densely innervated. Free protein gene product 9.5-immunoreactive fibres were less numerous but were present in all synovia examined, and in many cases these extended to the intimal layer. Neuropeptide immunostaining was predominantly found in perivascular networks. Fibres immunoreactive for the C-flanking peptide of neuropeptide Y were exclusively located around blood vessels whereas free fibres were immunoreactive for substance P or calcitonin gene-related peptide. As with free protein gene product 9.5-immunoreactive fibres, fibres expressing substance P or calcitonin gene-related peptide immunoreactivity were often seen in the intimal cell layer. In rheumatoid arthritis a similar innervation to that seen in normal synovium was apparent in the deep tissue but fibres immunoreactive for protein gene product 9.5, the C-flanking peptide of neuropeptide Y, substance P or calcitonin gene-related peptide were not visible in the more superficial tissues or the intimal cell layer. In addition, immunostaining of neuropeptides in the deep tissue was weaker in the diseased tissues than in normal controls. The data unequivocally demonstrate that synovial tissues are richly innervated and confirm the presence of both sensory and sympathetic nerves. The absence of nerves which innervate the superficial synovium in rheumatoid arthritis might suggest that there is increased release of substance P, calcitonin gene-related peptide and the C-flanking peptide of neuropeptide Y, reducing the stores in the nerves to levels below that detectable by immunocytochemistry. However, since protein gene product 9.5-immunoreactive nerves were not seen in the inflamed tissue it is probable that synovial growth outflanks neural growth and consequently as the disease progresses neural structures become restricted to deeper tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Short-rib-polydactyly syndrome type Verma-Naumoff-Le Marec in a fetus with histological hallmarks of type Saldino-Noonan but lacking internal organ abnormalities.

Up to seven short-rib-polydactyly (SRP) syndromes have been identified so far with marked clinical and pathological overlap. We describe a 32-week-old, nonhydropic male fetus with thoracic "dysplasia," short limbs, and unilateral postaxial polydactyly. All internal organs were normally developed, including the central nervous system. The external genitalia were unambiguously male, in accordance with a 46,XY karyotype. Radiological signs most closely resembled those of SRP, type Le Marec, though histology of the femoral physeal growth zone was consistent with the Saldino-Noonan type. The remarkable lack of visceral anomalies in conjunction with the radiological and histological findings further adds to the phenotypic spectrum of the SRP syndromes. The histological analysis in this case supports a close relationship between types Saldino-Noonan and Verma-Naumoff-Le Marec.

Review of the biological response to a novel bone cement containing poly(ethyl methacrylate) and n-butyl methacrylate.

This review describes work published independently elsewhere in which the biological reactions to poly(ethyl methacrylate) n-butyl methacrylate (PEMBMA) have been studied. This material has been compared throughout with conventional poly(methyl methacrylate) (PMMA). Butyl methacrylate monomer used in PEMBMA was slightly less toxic than methyl methacrylate monomer used in PMMA when injected intraperitoneally in mice. No differences in cardiorespiratory effects were found between n-butyl and methyl monomer infused intravenously into anaesthetized rabbits. The tissue reaction to the beaded polymers of poly(methyl methacrylate) and poly(ethyl methacrylate) implanted subcutaneously was identical. The surface appearance of the two materials differed significantly when viewed by scanning electron microscopy, showing a series of elevations resembling tightly packed spheres in the case of PMMA, but a smooth surface with only occasional smooth elevations in the case of PEMBMA. Intramuscular implantation showed more fibrous tissue and tissue damage in relation to PMMA cured in situ compared with PEMBMA and there was more bone necrosis and a thicker fibrous tissue layer adjacent to PMMA than PEMBMA when cured intraosseously.

Cancellous bone repair using bovine trabecular bone matrix particulates.

At 5 and 15 weeks post-surgery, biomechanical and histological analyses of cancellous bone defects filled with the bovine trabecular bone matrix (BBM) and hydroxyapatite (Hap) particulates of dimensions 106-150 microm were investigated. It was observed that at 5 weeks post-surgery the stiffness properties of the BBM filled defects were significantly higher than those observed in the Hap filled defects (p < 0.01) but comparable to those recorded in intact cancellous bone from the same anatomical position. Histologically, no significant differences were observed in the percentage of new bone contact with the particles. The biomechanical properties of the Hap filled defects mirrored those in intact cancellous bone only at 15 weeks post-surgery. BBM particles thus appeared to accelerate the early healing of osteotomies. It is therefore suggested that particles of this bioceramic be the subject of intense research for more usage in both periodontal osseous defects and orthopaedic fractures.

Hydrogels as an interface between bone and an implant.

The use of fully hydrated hydrogels in the body has been well established. The forces a hydrogel generates on swelling when it is placed in a constrained space were investigated with a view to providing a mechanism for fixing a prosthesis in the intramedullary cavity. A cross-linked poly(2-hydroxyethyl methacrylate) [p(HEMA)] hydrogel was investigated as a potential material. In vitro mechanical tests were carried out to determine the stresses generated in the p(HEMA) when it was placed in water and not allowed to swell. Pull out loads of up to 375 N indicated that the system could be used successfully in vivo. Consequently, the material was placed intraosseously at two sites in a rabbit animal model, in the mid-shaft (diaphysis) and the lower end (metaphysis) of the femur. Histological examination showed there was no adverse bone response; bone was growing from the endosteal surface up to and into the hydrogel in the diaphyseal implants and surrounded the hydrogel in the metaphysis. As a result of the shape and size variations in the rabbit femur, in vivo mechanical tests were found to give lower values than those obtained in vitro.

Assessment of the role of GM-CSF in the cellular transformation and the development of erosive lesions around orthopaedic implants.

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a hematopoietic growth factor with a regulatory effect on the transformation of immature macrophages into multinucleated giant cells (MNGC) that exhibit phenotypic and functional characteristics of osteoclasts (OC). The authors analyzed the bone implant interface membranes harvested from 15 patients with failed total joint replacements for the production and tissue distribution of GM-CSF and interleukin-1 (IL-1). Immunohistology and liquid culture were employed to assess the contribution of these factors in the recruitment of macrophages and the development of bone resorbing MNGC at these sites. This process has been implicated in osteoclastic bone resorption, bone, and bone marrow necrosis adjacent to orthopaedic implants. Histologic assessment of the interface indicated the presence of granuloma and a variable number of MNGC in 11 cases. Four cases showed sites of intramembranous formation of osteoid and mineralized bone that was accompanied by normal bone marrow in two cases. Granulocyte-macrophage colony stimulating factor was expressed by a distinct subset of phagocytic macrophages in the lining layer on the implant side. interleukin-1-positive cells outnumbered those stained for GM-CSF. Stimulation of cultured cells with prosthetic metal particulate material showed marked similarity in the expression of these cytokines to cultures treated with lipopolysaccharide (LPS) or phytohemagglutinin (PHA). The induction of GM-CSF production in the lining layer where small MNGC develop indicates that these cells differentiate locally following the phagocytosis of particulate wear debris. In conclusion, GM-CSF promotes the proliferation and early stages of fusion and development of MNGC responsible for osteolysis at these sites. These results also highlight the capacity of the interface to display both osteogenic and inflammatory characteristics. Collectively, the findings suggest that the local bone marrow could participate in the development of the interface as a source of myeloid cells in addition to the capacity of marrow stroma to generate various osteogenic cells essential for the ingrowth of bone into prosthetic implants.

Histomorphometry of bone.

This review of the histomorphometry of bone outlines methods of biopsy and processing of specimens in the laboratory, the basic principles of morphometry, and the measurements made in order to obtain estimates of the proportional volumes and surfaces occupied by different components of bone. Variability such as that between methods, observers and laboratories is discussed and a brief outline of automatic and semiautomatic methods of image analysis also given.

Remodelling of subchondral bone in osteoarthritis: a histomorphometric study.

AIMS: To determine whether remodelling of subchondral bone in osteoarthritis is related to anatomical and mechanical factors operating on the joint surface. METHODS: Ten femoral heads were examined. Patients due to have the femoral head removed because of osteoarthritis were given a double tetracycline label before surgery. The specimens were divided in three roughly equal parts, corresponding to the medial, central (weight bearing), and lateral aspects of the femoral heads. Undecalcified methylmethacrylate embedded sections were prepared. The subchondral bone was assessed for appositional bone formation by ultraviolet light microscopy, for resorptive activity by osteoclast count, and for trabecular bone volume by semiautomatic computerised image analysis. Appositional bone rate was also correlated with the presence or absence of overlying cartilage. RESULTS: Bone density was reduced in the medial aspect of the femoral heads but there was no significant difference between the appositional bone rate or the resorptive activity in the three areas. Nor was there any correlation between appositional bone rate in the subchondral bone and the presence or absence of overlying cartilage. CONCLUSION: Remodelling of subchondral bone in osteoarthritis, at least in the short term, is fairly constant and is not related to weight bearing.

Immunohistochemical analysis of mononuclear cell subsets in inflammatory and non-inflammatory myopathies.

Immunohistochemical procedures were used to analyse the subpopulations of mononuclear cells in muscle biopsies from 24 patients with polymyositis. The character of the cellular infiltrate was similar at the perivascular, perimysial, and endomysial sites, with cytotoxic-suppressor T lymphocytes (T8+) and macrophages being the dominant elements. Helper T lymphocytes (T4+) and B lymphocytes were present in smaller numbers. A control series of 17 muscle biopsies from normal subjects and patients with non-inflammatory myopathies and neurogenic conditions was also studied: the numbers of mononuclear cells present were much smaller than in polymyositis, but the ratio of T4:T8 lymphocytes was similar to that found in biopsies affected by polymyositis. We conclude that both cytotoxic-suppressor T lymphocytes and macrophages are important in the pathogenesis of inflammatory myopathy.

Yersinia virulence: more than a plasmid.

The genus Yersinia is composed of 11 species, three of which are pathogenic in humans. The three pathogens, Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis, cause a broad spectrum of disease ranging from pneumonic plague to acute gastroenteritis. Each of the three requires a large, well-defined plasmid for full virulence, as well as many chromosomally encoded virulence factors (CEVF). This review will describe these CEVF and their roles in virulence. In addition, a possible model for key events in Y. enterocolitica pathogenesis is described based on information revealed by analysis of several of the CEVF.

Differential expression of transforming growth factor-alpha and macrophage colony-stimulating factor/colony-stimulating factor-1R (c-fins) by multinucleated giant cells involved in pathological bone resorption at the site of orthopaedic implants.

The immunologic response to prosthetic biomaterial particles is characterized by macrophage-rich inflammatory infiltrate, formation of multinucleated giant cells, and aseptic loosening at the site of arthroplasty. We investigated the in vivo expression and tissue distribution of transforming growth factor alpha, macrophage colony-stimulating factor, and the receptor for colony-stimulating factor-1 at the site of bone erosion in patients with clinically failed orthopaedic implants (n = 30). The expression was further compared with that detected in the inflamed synovial membranes from patients with rheumatoid arthritis or osteoarthritis (n = 15) and one patient with osteoclastoma (giant cell tumour of bone). Immunostaining of the tissue demonstrated positivity for transforming growth factor alpha within the inflammatory macrophage and multinucleated giant cell infiltrate in the diseased synovial membrane and the bone-implant interface. A comparative analysis between the synovium and retrieval interface membranes (pseudosynovium) revealed a high level of expression of transforming growth factor alpha, with intense membrane staining on multinucleated giant cells in all failed arthroplasties with pseudosynovium. In addition, the frequency, antigenic phenotype, and pattern of transforming growth factor alpha expression on multinucleated giant cells in the interface were markedly similar to those observed for multinucleated giant cells in osteoclastoma. Multinucleated giant cells within the interface lacked the expression of macrophage colony-stimulating factor and colony-stimulating factor-1 receptor, whereas those at the bone surfaces exhibited strong immunoreactivity. The predominant expression of transforming growth factor alpha by multinucleated giant cells in the bone-implant interface and its similarity to osteoclastoma highlight the importance of assessing transforming growth factor alpha as a possible contributor to the development of bone-resorbing giant cells at the site of failed orthopaedic implants.

Bone formation and bone resorption in failed total joint arthroplasties: histomorphometric analysis with histochemical and immunohistochemical technique.

To elucidate the reactions of bone around aseptically loosened total joint arthroplasties, 24 interface tissues with adjacent bone were obtained in 17 revision operations (11 hips and six knees). The morphology of the bone surface next to the interface membrane was investigated with histochemical and immunohistochemical techniques and then histomorphometrically analysed. One-third of the total bone surface. 32.69 +/- 5.16% (mean +/- SE) (n = 24), showed positive alkaline phosphatase activity. The bone surface in contact with the cells positive for CD11b (a macrophage marker) amounted to 19.33 +/- 5.16% (n = 24). The proportion of the osteoclastic bone resorption estimated by vitronectin receptor expression was 7.67 +/- 1.82% (n = 21). Tissues retrieved from the sites where radiographic evidence of osteolysis was present (n = 12) had a significantly larger extent of the bone surface in contact with CD11b-positive cells than did the tissues from areas without osteolysis (n = 12, p = 0.0067, Mann-Whitney U test), whereas no significant difference was observed in the extent of osteoclastic bone resorption. These data demonstrate that active bone formation, regarded as a repair process, is the most common feature even in revised cases. They also highlight the role played by macrophages, not as cells producing inflammatory mediators that could activate osteoclasts, but as cells primarily responsible for the bone loss in osteolytic lesions.

Lymphocytes, polymorphonuclear leukocytes, macrophages and platelets in synovium involved by rheumatoid arthritis. A study with monoclonal antibodies.

The inflammatory cell infiltrate involving synovial tissues from joints affected by rheumatoid arthritis (RA)++ has been contrasted with that present in synovium removed from joints involved by previous trauma (T) or osteoarthritis (OA). Cell deployment has been mapped by immunohistochemistry using monoclonal antibodies which recognise epitopes characterising T and B cells, polymorphonuclear leukocytes, mononuclear phagocytes and platelets. Mononuclear phagocytes were the most consistent feature of the rheumatoid inflammatory cell exudate and were present, particularly in the synovial layer, in all OA/T samples. The synovial cells lacked the C3b complement receptor, CR1, but expressed CR3, the receptor for C3bi. In rheumatoid synovium, interdigitating cells were difficult to identify but cells of dendritic morphology bore at least one macrophage epitope. T cells far out-numbered B cells and generally lacked the IL-2 receptor which is an indicator of T cell activation. Care is required in the estimation of the T helper/inducer (TH) T suppressor/cytotoxic (Ts) ratio. Polymorphonuclear leukocytes were demonstrated around vessels and near the synovial intimal cell layer suggesting rapid tissue transit. Extravascular platelets were sparse. Follicular dendritic cells were defined by their central location in lymphoid follicles and strong expression of CR1 receptors. HLA-DR expression was widespread except on endothelial cells.

The composition of normal and osteoarthritic articular cartilage from human knee joints. With special reference to unicompartmental replacement and osteotomy of the knee.

UNLABELLED: The articular cartilage from nineteen osteoarthritic and fourteen normal control adult human knee joints was analyzed for changes in water content, proteoglycan composition and structure, glycosaminoglycan synthesis rates, and cell content. We found no significant differences between visually intact cartilage from osteoarthritic knee joints and cartilage from control joints for any of the parameters studied. In osteoarthritic specimens in which the cartilage surface was not intact the biochemical changes depended on the degree of fibrillation. Surface-fibrillated specimens had a higher water content in the surface layers but no change in the content or synthesis rate of glycosaminoglycan. Deeply fibrillated cartilage, however, had an increased water content through its full depth, and there was a decrease in both the rate of synthesis and the content of glycosaminoglycans. CLINICAL RELEVANCE: The results of this study suggest that degenerative changes in osteoarthritic knees are focal in origin and that corrective osteotomy or unicompartmental joint replacement might be rational procedures for knees in which the cartilage in all of one compartment is visually intact.