Virus inactivation in bone tissue transplants (femoral heads) by moist heat with the 'Marburg bone bank system'

Several virus inactivation procedures like heat treatment, gamma irradiation and chemical sterilization are used to increase the safety of bone tissue transplants. In this study we present data on the virus-inactivating effect of heat disinfection on human femoral heads, using the Marburg bone bank system 'Lobator sd-2'. Three enveloped viruses (human immunodeficiency virus type 2 [HIV-2], bovine viral diarrhoea virus as a model for Hepatitis C virus [HCV], and the herpesvirus pseudorabies virus), and three non-enveloped viruses (hepatitis A virus, poliomyelitis virus, and bovine parvovirus) were investigated.In a model system the central part of human femoral heads was contaminated with the respective cell-free virus suspension, establishing a direct contact between virus and native bone tissue. The core temperature in the femoral heads during the sterilization process was determined in additional model experiments. A temperature of 82.5 degrees C, given by the manufacturer as the effective temperature for virus inactivation, was maintained for at least 15 min in decartilaged femoral heads with a diameter of < or = 56 mm. Heat treatment using the Lobator sd-2 inactivated all viruses in human femoral heads below the detection limit (at least by a factor of > or =4 log(10)).By combining a well-focussed anamnesis of the donors and serological testing for relevant infection markers (anti-HIV-1/2, HBsAg, anti-HBcore, anti-HCV, TPHA) with heat treatment of femoral heads in the Lobator sd-2 system, a high safety level is achieved. To further increase virus safety of cadaveric bone transplants, it is recommended that multi-organ donors are tested by nucleic acid testing (i.e. polymerase chain reaction) for HIV, HBV and HCV genome.     

The Prevalence of Human T-Lymphotropic Virus Infection among Blood Donors in Southeast China, 2004-2013

Background

The human T-lymphotropic virus type 1 (HTLV-1) which is associated with the diseases of adult T-cell leukemia/lymphoma, HTLV-1 associated myelopathy / tropical spastic paraparesis (HAM/TSP) and HTLV-associated uveitis, can cause transfusion-transmitted infections. Although HTLV screening of blood donors was already routinely performed in developed countries, little is know about the HTLV prevalence among blood donors in developing countries which do not perform HTLV screening, such as China.

Objectives &Aims

To systematically characterize the prevalence of HTLV infection among bloods in southeast China.

Methods

A 10-year survey for HTLV prevalence in blood donors was performed in Xiamen, southeast China, during 2004-2013. The HTLV-1/2 of blood donations were screened by enzyme-linked immunosorbent assay, following with confirmation by western blot assay and 9nucleic acid testing. The HTLV-1 prevalences in donors from different cities were calculated. Viral sequences derived from identified HTLV-positive cases were sequenced and analyzed.

Results

Among 253,855 blood donors, 43 were confirmed to be seropositive for HTLV-1 (16.9 per 100,000 95% CI: 12.3-22.8) and none HTLV-2 infection was found. The HTLV-1 prevalence varied significantly in donors from different cities. Donors from cities in Fujian province (24.3 per 100,000, 95%CI: 17.4-33.1) had a significantly higher (p=0.001) HTLV-1 seroprevalence than those who were born in non-Fujian cities (3.4 per 100,000, 95%CI: 0.7-9.8). Among nine cities in Fujian province, the highest prevalence was found in blood donors from Ningde (171.3 per 100,000, 95%CI: 91.3-292.8) which is a coastal city in the northeast of Fujian. Molecular characterization of viral sequences from 27 HTLV-1 carriers revealed 25 were Transcontinental subtype of genotype A and 2 were Japanese subtype of genotype A. Interestingly, 12 of 25 Transcontinental subtype sequences harbored a characteristic L55P mutation in viral gp46 protein, which was only presented in the Transcontinental subtype sequences from Japan and Taiwan but not in that from other countries.

Conclusions

Although China is considered to be a non-endemic region for HTLV, the HTLV-1 prevalence in blood donors is significantly higher in Fujian province, southeast China. A higher prevalence of HTLV-1 in the Fujian may be attributed to endemic foci in the city of Ningde.     

Post-operative infection with fresh frozen allograft: reported outcomes of a hospital-based bone bank over 14 years

Femoral head bone allografts have traditionally been used to provide mechanical stability to areas of bony deficiency, or for its osteoinductive and osteoconductive properties. Concerns have been raised over increased infection rates following the use of fresh-frozen graft tissue. This retrospective study aims to investigate the outcomes of fresh frozen femoral heads kept in a regulated, non-commercial bone bank at a university teaching hospital.The local bone bank database was used to identify released femoral heads during a 14 year study period (September 1999–December 2013) whereby a retrospective review of patient records was undertaken to determine clinical outcome. During the observed study period, 427 femoral heads were released from cold storage. Of these, 270 femoral heads had a mean follow-up of 347 days. 157 femoral heads were excluded due to insufficient follow-up data (n = 132) or discarded due to breaks in the cold chain prior to use (n = 25). Of the 270 included femoral heads, 231 (85.6 %) had no reported complications with good graft incorporation. In the remaining 39 with reported complications, only 5 (2.6 %) developed a postoperative infection. Our findings suggest that the use of fresh frozen allograft does not materially increase the risk of post-operative bacterial infection. Our reported post-operative infection rates are comparable with infection rates of other similar studies on fresh frozen allograft use.     

Highly enhanced expression of CD70 on human T-lymphotropic virus type 1-carrying T-cell lines and adult T-cell leukemia cells

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4(+) T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4(+) T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.     

Characterization and sequencing of prototypic human T-lymphotropic virus type 1 (HTLV-1) from an HTLV-1/2 seroindeterminate patient

Serological screening for human T-lymphotropic virus type 1 (HTLV-1) parallels the standard screening process for human immunodeficiency virus (HIV), in which samples found positive by enzyme-linked immunosorbent assay (ELISA) are confirmed with a modified Western blot procedure. There are a significant number of cases in which HTLV-1/2 ELISA-positive specimens demonstrate an incomplete banding pattern on this Western blot. Individuals providing these atypical antibody responses are categorized as seroindeterminate for HTLV-1/2. Although HTLV-1 genomic sequences are readily detectable in the peripheral blood lymphocytes (PBL) of seropositive individuals, previous studies have repeatedly demonstrated that PBL from the vast majority of HTLV-1/2 seroindeterminate individuals are PCR negative for HTLV-1. As a result, identification of the agent responsible for this indeterminate reactivity has been of interest. We have generated an HTLV-1-positive B-cell line (SI-1 B) from one of these seroindeterminate individuals. Previous screening for HTLV-1 in PBL from this patient had been routinely negative by primary PCR; however, HTLV-1 tax had been periodically detected by nested PCR. DNA sequence data generated with genomic DNA from the SI-1 B cell line and HTLV-1-specific primers demonstrated the presence of a full-length viral genome with >97% homology to the Cosmopolitan form of HTLV-1. A 12-bp deletion was identified in the 3'-gag/5'-prot region, which would predict translation of altered or nonfunctional proteins from these genes. We propose that this HTLV-1/2-seroindeterminate patient is infected with a prototypic form of HTLV-1 at an extremely low viral load and that this finding may explain HTLV-1/2 seroindeterminate reactivity in at least a subset of these individuals.     

Assessment of a closed wash system developed for processing living donor femoral heads

NHS Blood and Transplant Tissue and Eye Services (TES) and Scottish National Blood Transfusion Services Tissues and Cells Directorate (TCD) currently bank whole, frozen femoral head bone from living donors who are undergoing primary hip replacement surgery. When required, the bone is issued to a surgeon still frozen on dry ice (? 79 °C). Consequently, the femoral head bone is not processed, is not sterilised and at the time of issue, it contains donor blood, bone marrow and associated cells. We have previously shown that, cut, shaped and washed bone from deceased donors can be processed to remove up to 99.9% of blood, bone marrow and associated cells (Eagle et al. 2015). However, cut and shaped bone is not suitable for some orthopaedic procedures and some orthopaedic surgeons do not wish to use irradiated bone; therefore in this report, a method has been developed in which whole femoral heads can be washed to remove donor blood and bone marrow components. Processing results in excess of 99% bone marrow component removal—soluble protein, haemoglobin and DNA; the procedure is performed inside a closed system, thereby eliminating the need for terminal sterilisation by irradiation. In addition, uniaxial testing demonstrated no difference in compressive strength between washed and unwashed bone. We suggest that this washed bone may be capable of improving incorporation after grafting without disturbing biomechanical properties of the graft.     

Improving efficiency of a regional stand alone bone bank

The introduction of a stand-alone Bone Bank in our Regional Orthopaedic Hospital has improved the availability of femoral head allograft. Benninger et al. (Bone Joint J 96-B:1307–1311, 2014), demonstrated their institutions bank to be cost effective despite a 30 % discard rate for harvested allograft. We sought to audit our own discard rates and subsequent cost-effectiveness of our bone bank. Donor recruitment. Before approaching a potential donor, our establishment’s nurse specialists review their clinical notes and biochemical laboratory results, available on a regional Electronic Care Records. They view femoral head architecture on radiographs against set criteria, Patient Archive and Communication system (SECTRA, Sweden). In total 1383 femoral heads were harvested, 247 were discarded giving an overall rate of 17.9 %. The most common reasons for discard of harvested graft was a positive microbiology/bacteriology result, n = 96 (38.9 %). After a rise in discard rates in 2007, we have steadily reduced our discard rates since 2006/2007 (28.2 %), 2008/2009 (17 %), 2010/2011 (14.8 %), and finally to 10.3 % in 2012/2013. In the current financial year, our cost to harvest, test, store and release a femoral head is £610. With a structured donor recruitment process and unique pre-operative radiographic analysis we have successfully reduced our discard rates bi-annually making our bone bank increasingly cost-effective.     

Coinfection by Strongyloides stercoralis in blood donors infected with human T-cell leukemia/lymphoma virus type 1 in São Paulo City, Brazil

The frequency of coinfection with Strongyloides stercoralis and human T-cell leukemia/lymphoma virus type 1 (HTML-1) was determined in 91 blood donors examined at the blood bank of a large hospital in S?o Paulo city, Brazil. As control group 61 individuals, not infected by HTLV-1, were submitted to the same techniques for the diagnosis of S. stercoralis infection. In HTLV-1 infected patients the frequency of S. stercoralis infection was 12.1%; on the other hand, the control group showed a frequency significantly lower of S. stercoralis infection (1.6%), suggesting that HTLV-1 patients should be considered as a high risk group for strongyloidiasis in S?o Paulo city.     

HTLV-1病毒蛋白Tax和HBZ协同促进成人T细胞白血病发生的机制

人类T细胞白血病1型病毒(Human T-cell leukemia virus type 1,HTLV-1)是与人类疾病发生密切相关的逆转录病毒。该病毒的感染可引起成人T细胞白血病(Adult T-cell leukemia,ATL)等多种疾病的发生。Tax和HBZ(HTLV-1 basic zipper protein)是由HTLV-1前病毒编码的两个关键病毒蛋白,它们被认为在HTLV-1病毒的复制、生存和致癌过程中发挥了至关重要的作用。本文就Tax和HBZ的蛋白结构以及它们在调控病毒转录、细胞生长和凋亡、病毒潜伏期,最终协同促进成人T细胞白血病发生过程中发挥的作用作一综述。     

Prevalence and evolutionary analyses of human T-cell lymphotropic virus in Guangdong province,China: Transcontinental and Japanese subtype lineages dominate the prevalence

To systematically characterize the prevalence and evolution of human T-cell lymphotropic virus (HTLV) infection among voluntary blood donors (BDs) in Guangdong province, China. A three-year survey for HTLV epidemiology among BDs was performed in Guangdong during 2016–2018. Anti-HTLV-1/2 was screened by ELISA and ECLIA, and subsequently confirmed by western blot (WB) and nucleic acid testing (NAT). The prevalence of HTLV in donors from different cities was calculated. The identified HTLV-positive cases were phylogenetically genotyped and analyzed in a Bayesian phylogenetic framework. Among 3,262,271 BDs, 59 were confirmed positive for HTLV-1 (1.81 per 100,000) and no HTLV-2 infection was found. The prevalence of HTLV-1 varied significantly among 21 cities in Guangdong province, China. The highest prevalence was found in donors from Shanwei (13.94 per 100,000), which is a coastal city in eastern Guangdong. Viral genomic sequences genotyped from 55 HTLV-1 carriers showed that 39 were transcontinental subtype and 16 were Japanese subtype. Specially, 13 out of 39 transcontinental subtype sequences were characterized with L55P mutation and 21 out of 55 sequences were characterized with L19F mutation in viral gp46 protein. The L55P mutation seemed be specific to eastern Asia since it only presented in the sequences from Japan, mainland China, and Taiwan. Phylogenetic analysis of gp46 gene shows that HTLV-1a may have been introduced to Guangdong through four different introduction events and formed major transmission clusters: clades I(13,602 years ago), II(16, 010 years ago), III(15,639 years ago) and IV(16,517 years ago). In general, Guangdong is considered to be a low-prevalence region for HTLV-1 infection, but the prevalence is significantly higher in Shanwei city. Transcontinental and Japanese subtype lineages dominate the prevalence in Guangdong. In terms of blood safety, HTLV antibody screening for first-time blood donors can effectively reduce the risk of HTLV transmission.     

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Chile

The Tissue Banking Project in Chile started as an idea in 1996. Before 1996 in Chile there were only a few small bone banks working with their own standards of quality. The first tissue bank (LPTR) was established in 1998, with the technical and financial support of the IAEA. Since 2001, the laboratory began to produce tissues for clinical use, starting with the processing of 6 amniotic membranes, 2 femoral heads and 19 batches of pig skin. In 2002, the laboratory began the processing of human skin. Five students from Chile have graduated from training courses carried out in Singapore and in Buenos Aires under the IAEA training program since 1998. The amount of tissues produced and sterilized using ionizing radiation by the LPTR in the last years was 320,000 cm² of human skin, 553,600 cm² of pig skin, 5,400 cm² of amniotic membrane, 49 femoral heads, 3 large bones and 300 g of bovine bone. The patients treated with sterilized tissues produced by the LPTR were 200 deep burns treated with human skin and pig skin, 40 bone transplants from femoral heads, 77 ophthalmologic patients treated with amniotic membrane and 150 bovine bone transplants for dental treatments.     

Interactions between brain endothelial cells and human T-cell leukemia virus type 1-infected lymphocytes: mechanisms of viral entry into the central nervous system

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.     

Expression of glucose transporter 1 confers susceptibility to human T-cell leukemia virus envelope-mediated fusion

Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus identified and causes both adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-1-associated myelopathy, among other disorders. In vitro, HTLV-1 has an extremely broad host cell tropism in that it is capable of infecting most mammalian cell types, although at the same time viral titers remain relatively low. Despite years of study, only recently has a bona fide candidate cellular receptor, glucose transporter 1 (glut-1), been identified. Although glut-1 was shown to bind specifically to the ectodomain of HTLV-1 and HTLV-2 envelope glycoproteins, which was reversible with small interfering RNA directed against glut-1, cellular susceptibility to HTLV upon expression of glut-1 was not established. Here we show that expression of glut-1 in relatively resistant MDBK cells conferred increased susceptibility to both HTLV-1- and HTLV-2-pseudotyped particles. glut-1 also markedly increased syncytium formation in MDBK cells after exposure to HTLV-1. Another assay also demonstrated HTLV-1 envelope-cell fusion in the presence of glut-1. Taken together, these results provide additional evidence that glut-1 is a receptor for HTLV.     

Stresses in cement mantles of hip replacements: effect of femoral implant sizes,body mass index and bone quality

The effects of femoral prosthetic heads of diameters 22 and 28 mm were investigated on the stability of reconstructed hemi-pelves with cement mantles of thicknesses 1–4 mm and different bone qualities. Materialise medical imaging package and I-Deas finite element (FE) software were used to create accurate geometry of a hemi-pelvis from CT-scan images. Our FE results show an increase in cement mantle stresses associated with the larger femoral head. When a 22 mm femoral head is used on acetabulae of diameters 56 mm and above, the probability of survivorship can be increased by creating a cement mantle of at least 1 mm thick. However, when a 28 mm femoral head is used, a cement mantle thickness of at least 4 mm is needed. Poor bone quality resulted in an average 45% increase in the tensile stresses of the cement mantles, indicating resulting poor survivorship rate.     

Bone bank service in Odense, Denmark. Audit of the first ten years with bone banking at the Department of Orthopaedics, Odense University Hospital

There has been an increase in the demand for allograft bone in recentyears. The Odense University Hospital bone bank has been in function since1990,and this paper outlines our results during the 10 year period 1990–1999.Potential donors were screened by contemporary banking techniques which includea social history, donor serum tests for HIV, hepatitis B and C, and graftmicrobiology. The bones were stored at –80 °C. No typeofsecondary sterilisation was made. 423 femoral heads were approved and donatedto300 patients,1–6 heads/operation. The allografts have been used mainly toreconstruct defects at revision hip arthroplasty (34%), and for fracturesurgery(24%). 7 % of all transplanted patients were reoperated because of infection.Inthe hip revision group the infection rate was 4 %. There were no cases ofdisease transmission. During the 10 year period there was a change in theclinical use of the allografts. In the first years the allografts were mainlyused for spinal fusion surgery, but today the majority are used in hip revisionand fracture surgery. The clinical results correspond to those reported inlarger international series.