Paula A. Revell

A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules

Phase pure hydroxyapatite (HA) and a 0.8 wt % silicon substituted hydroxyapatite (SiHA) were prepared by aqueous precipitation methods. Both HA and SiHA were processed into granules 0.5–1.0 mm in diameter and sintered at 1200 °C for 2 h. The sintered granules underwent full structural characterization, prior to implantation into the femoral condyle of New Zealand White rabbits for a period of 23 days. The results show that both the HA and SiHA granules were well accepted by the host tissue, with no presence of any inflammatory cells. New bone formation was observed directly on the surfaces and in the spaces between both HA and SiHA granular implants. The quantitative histomorphometry results indicate that the percentage of bone ingrowth for SiHA (37.5%±5.9) was significantly greater than that for phase pure HA (22.0%±6.5), in addition the percentage of bone/implant coverage was significantly greater for SiHA (59.8%±7.3) compared to HA (47.1%±3.6). These findings indicate that the early in vivo bioactivity of hydroxyapatite was significantly improved with the incorporation of silicate ions into the HA structure, making SiHA an attractive alternative to conventional HA materials for use as bone substitute ceramics.

Sensitivity to titanium. A cause of implant failure

Tissues from five patients who underwent revision operations for failed total hip replacements were found to contain large quantities of particulate titanium. In four cases this metal must have come from titanium alloy screws used to fix the acetabular component; in the fifth case it may also have originated from a titanium alloy femoral head. Monoclonal antibody labelling showed abundant macrophages and T-lymphocytes, in the absence of B-lymphocytes, suggesting sensitisation to titanium. Skin patch testing with dilute solutions of titanium salts gave negative results in all five patients. However, two of them had a positive skin test to a titanium-containing ointment.

Observations upon the interface between bone and polymethylmethacrylate cement

The occurrence of a radiolucent line at the interface of bone and cement in total joint prostheses is a frequently observed, although little understood, phenomenon. Because of an operative technique utilised in instances of bone loss, we have, within a single implant mass used in each of a series of 18 total knee replacements, been able to observe two separate interfaces, one between bone and cement and the other between bone and cobalt chrome. The average period of observation was 32 months. All of the knees except one demonstrated a lucency at the bone-cement interface; only one of the knees had a similar lucency at the bone-CoCr interface. One of the knees was studied histologically. In the light of the universal observation of macrophages at bone-cement interfaces and the recent finding that osteoclasts are derived from macrophages, these observations are significant in relation to the aetiology of bone-cement lucencies.

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)

The synovial membrane in osteoarthritis: a histological study including the characterisation of the cellular infiltrate present in inflammatory osteoarthritis using monoclonal antibodies.

Inflammatory infiltration of the synovial membrane has been described in a proportion of cases of osteoarthritis (OA). Using conventional histology, lymphoid follicles, diffuse fibrosis, and perivascular fibrosis were shown to be present to a significantly greater extent and in more synovial membranes in osteoarthritis than in those cases where there was a mechanical or traumatic background to the joint disease. Calcium pyrophosphate dihydrate crystals (five patients) and detritus fragments of bone and cartilage (seven patients) were present in small numbers of the total cases of OA (38) studied. Neither of these features was related to the presence of an inflammatory infiltrate. Examination of 20 osteoarthritic synovial membranes using monoclonal antibodies showed the presence of lymphoid follicles containing T helper and T suppressor lymphocytes, B lymphocytes, and macrophages expressing HLA-DR in five cases. The T helper:suppressor ratio varied between 1:1 and 2.5:1 in these follicles. In addition, half of the OA samples, including these five cases, showed the presence of a diffuse cellular infiltrate containing T and B lymphocytes and macrophages, which were HLA-DR positive. Granulocytes were present in this diffuse infiltrate in those cases containing lymphoid follicles. The results confirm the presence of an inflammatory form of osteoarthritis but also show that the proportions of lymphoid cells are not the same as those considered to be typical of rheumatoid arthritis.

Vaccine-Acquired Rotavirus in Infants with Severe Combined Immunodeficiency

Live pentavalent human-bovine reassortant rotavirus vaccine is recommended in the United States for routine immunization of infants. We describe three infants, two with failure to thrive, who had dehydration and diarrhea within 1 month after their first or second rotavirus immunization and subsequently received a diagnosis of severe combined immunodeficiency. Rotavirus was detected, by means of reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay, in stool specimens obtained from all three infants, and gene-sequence analysis revealed the presence of vaccine rotavirus. These infections raise concerns regarding the safety of rotavirus vaccine in severely immunocompromised patients.

A chromosomally encoded regulator is required for expression of the Yersinia enterocolitica inv gene and for virulence

The primary invasion factor of Yersinia enterocolitica, invasin, is encoded by inv. inv expression is regulated in response to pH, growth phase and temperature. In vitro, inv is maximally expressed at 26°C, pH 8.0, or 37°C, pH 5.5, in early stationary phase. At 37°C, pH 8.0, inv is weakly expressed. To identify which gene(s) are required for inv regulation, we screened for transposon insertions that decreased expression of an inv–′phoA chromosomal reporter at 26°C. Of 30 000 mutants screened, two were identified that had negligible inv expression in all conditions tested. Both of these independent mutants had an insertion into the same gene, designated rovA (regulator of virulence). RovA has 77% amino acid identity to the Salmonella typhimurium transcriptional regulator SlyA. Complementation with the wild‐type rovA allele restores wild‐type inv expression as monitored by Western blot analysis, tissue culture invasion assay and alkaline phosphatase assay. There is also a significant decrease in invasin levels in bacteria recovered from mice infected with the rovA mutant; therefore, RovA regulates inv expression in vivo as well as in vitro. In the mouse infection model, an inv mutant has a wild‐type LD50, even though the kinetics of infection is changed. In contrast, the rovA mutant has altered kinetics, as well as a 70‐fold increase in the LD50 compared with wild type. Furthermore, because the rovA mutant is attenuated in the mouse model, this suggests that RovA regulates other virulence factors in addition to inv. Analysis of other proposed virulence factors such as Ail, YadA and the Yop proteins shows no regulatory role for RovA. The more severe animal phenotype combined with the lack of impact on known virulence genes aside from inv suggests RovA regulates potentially novel virulence genes of Y. enterocolitica during infection.

Granzyme B and the Downstream Granzymes C and/or F Are Important for Cytotoxic Lymphocyte Functions

Although the functions of granzyme A (GzmA) and GzmB are well-defined, a number of orphan granzymes of unknown function are also expressed in cytotoxic lymphocytes. Previously, we showed that a targeted loss-of-function mutation for GzmB was associated with reduced expression of several downstream orphan granzyme genes in the lymphokine-activated killer cell compartment. To determine whether this was caused by the retained phosphoglycerate kinase I gene promoter (PGK-neo) cassette in the GzmB gene, we retargeted the GzmB gene with a LoxP-flanked PGK-neo cassette, then removed the cassette in embryonic stem cells by transiently expressing Cre recombinase. Mice homozygous for the GzmB null mutation containing the PGK-neo cassette (GzmB−/−/+PGK-neo) displayed reduced expression of the closely linked GzmC and F genes in their MLR-derived CTLs and lymphokine-activated killer cells; removal of the PGK-neo cassette (GzmB−/−/ΔPGK-neo) restored the expression of both genes. Cytotoxic lymphocytes derived from mice with the retained PGK-neo cassette (GzmB−/−/+PGK-neo) had a more severe cytotoxic defect than those deficient for GzmB only (GzmB−/−/ΔPGK-neo). Similarly, GzmB−/−/+PGK-neo mice displayed a defect in the allogeneic clearance of P815 tumor cells, whereas GzmB−/−/ΔPGK-neo mice did not. These results suggest that the retained PGK-neo cassette in the GzmB gene causes a knockdown of GzmC and F expression, and also suggest that these granzymes are relevant for the function of cytotoxic lymphocytes in vitro and in vivo.

Daptomycin therapy for invasive Gram-positive bacterial infections in children.

Background: Clinical improvement is often delayed among children with invasive infections caused by multidrug resistant Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) despite use of standard antimicrobial therapy. Daptomycin, a bactericidal lipopeptide antibiotic, may prove useful for treatment of these infections in children, but clinical experience is lacking. Methods: Retrospective review of medical records of hospitalized children who received daptomycin for treatment of invasive Gram-positive bacterial infections at Children’s Medical Center Dallas from December 2003 to March 2007. Bacterial isolates were tested for susceptibility to daptomycin and characterized by pulsed-field gel electrophoresis and polymerase chain reaction for staphylococcal cassette chromosome mec A. Results: Sixteen children (10 male; median age, 6.5 years) received daptomycin. Fifteen (94%) children had invasive staphylococcal disease (14, MRSA, of which 13 were community-associated; 1, methicillin-susceptible S. aureus), and 1 had urinary tract infection caused by VRE. Twelve children with disseminated staphylococcal disease had bacteremia for 2–10 days despite therapy with 2 or more of the following: vancomycin, clindamycin, rifampin, aminoglycoside, or linezolid. The addition of daptomycin resulted in bacteriologic cure in 6 of 7 evaluable patients with persistent bacteremia. No adverse events were attributed to daptomycin. Overall, 14 patients improved and were discharged home, and 2 died of complications of their underlying medical conditions. Conclusions: The majority of patients demonstrated clinical improvement after addition of daptomycin to conventional antimicrobial therapy. Further studies are needed to assess the pharmacokinetics, pharmacodynamics, safety, and effectiveness of daptomycin in infants and children.

Comparison of Automated Nucleic Acid Extraction Methods with Manual Extraction

Automated nucleic acid extractors can improve workflow and decrease variability in the clinical laboratory. We evaluated Qiagen EZ1 (Valencia, CA) and bioMérieux (Durham, NC) easyMAG extractors compared with Qiagen manual extraction using targets and matrices commonly available in the clinical laboratory. Pooled samples were spiked with various organisms, serially diluted, and extracted in duplicate. The organisms/matrices were Bordetella pertussis/bronchoalveolar lavage, herpes simplex virus II/cerebrospinal fluid, coxsackievirus A9/cerebrospinal fluid, BK virus/plasma, and Mycoplasma pneumoniae/endotracheal tube samples. Extracts were amplified in duplicate using real-time PCR assays, and amplification of the target at a cycle threshold of 35 using the manual method was used for comparison. Amplification efficiency of nucleic acids extracted by automated methods was similar to that by the manual method except for a loss of efficiency for M. pneumoniae in endotracheal tube samples. The EZ1 viral kit 2.0 gave better results for coxsackievirus A9 than the EZ1 viral kit version 1.0. At the lowest limit of detection (past a cycle threshold of 35), the easyMAG was more likely to produce amplifiable nucleic acid than were either the EZ1 or manual extraction. Operational complexity, defined as the number of manipulations required to obtain an extracted sample, was the lowest for the easyMAG. The easyMAG was the most expensive of the methods, followed by the EZ1 kit and manual extraction.