Potential neoplastic evolution of Vero cells: in vivo and in vitro characterization.

Vero cell lines are extensively employed in viral vaccine manufacturing. Similarly to all established cells, mutations can occur during Vero cells in vitro amplification which can result in adverse features compromising their biological safety. To evaluate the potential neoplastic evolution of these cells, in vitro transformation test, gene expression analysis and karyotyping were compared among low- (127 and 139 passages) and high-passage (passage 194) cell lines, as well as transformed colonies (TCs). In vivo tumorigenicity was also tested to confirm preliminary in vitro data obtained for low passage lines and TCs. Moreover, Vero cells cultivated in foetal bovine serum-free medium and derived from TCs were analysed to investigate the influence of cultivation methods on tumorigenic evolution. Low-passage Vero developed TCs in soft agar, without showing any tumorigenic evolution when inoculated in the animal model. Karyotyping showed a hypo-diploid modal chromosome number and rearrangements with no difference among Vero cell line passages and TCs. These abnormalities were reported also in serum-free cultivated Vero. Gene expression revealed that high-passage Vero cells had several under-expressed and a few over-expressed genes compared to low-passage ones. Gene ontology revealed no significant enrichment of pathways related to oncogenic risk. These findings suggest that in vitro high passage, and not culture conditions, induces Vero transformation correlated to karyotype and gene expression alterations. These data, together with previous investigations reporting tumour induction in high-passage Vero cells, suggest the use of low-passage Vero cells or cell lines other than Vero to increase the safety of vaccine manufacturing.

Diagnostic delay and prognosis in primary central nervous system lymphoma compared with glioblastoma multiforme.

Glioblastoma multiforme (GBM) and primary central nervous system lymphoma (PCNSL) are malignant cerebral neoplasms associated with poor prognosis. Early diagnosis and subsequent planning of adequate treatment strategy are relevant to improve survival and reduce neurological deficit. Two groups of patients affected by GBM and PCNSL were compared to identify: (1) factors influencing the time necessary to obtain a correct diagnosis; (2) the influence of the interval time from clinical onset to diagnosis on the prognosis. Fifty-six patients (28 PCNSL and 28 GBM, 23 females and 33 males) referred to the same hospital setting were retrospectively evaluated. The mean age at diagnosis was 61 years. The two groups were comparable in terms of age, sex, clinical symptoms at onset and performance status. There was no relevant difference in time span from clinical onset to first neuroimaging examination, while time span from first neuroimaging to final morphological diagnosis was much longer in PCNSL patients (p = 0.008). Multivariate Cox regression analysis, including both PCNSL and GBM cases, showed a significant association of the overall survival with: time to diagnosis (HR 0.06), age at onset (HR 1.04). Our results show a significant diagnostic delay in PCNSL cases. Age at onset of disease and time to diagnosis emerge as clinical factors affecting overall survival in both groups. Stereotactic-guided biopsy should be chosen as routine method to early diagnose PCNSL. The clinical relevance of early diagnosis in GBM and PCNSL needs to be emphasized to maximize the overall survival in both neoplasms.

Allogeneic adipose tissue-derived mesenchymal stem cells in combination with platelet rich plasma are safe and effective in the therapy of superficial digital flexor tendonitis in the horse.

Overstrain tendonitis are common pathologies in the sport horses. Therapeutic approaches to tendon healing do not always result in a satisfactory anatomical and functional repair, and healed tendon is often characterized by functional impairment and high risk of reinjury. Recently, mesenchymal stem cells (MSCs) and platelet rich plasma (PRP) have been proposed as novel therapeutic treatments to improve the tendon repair process. MSCs are multipotent, easy to culture and being originated from adult donors do not pose ethical issues. To date, autologous MSCs have been investigated mainly in the treatment of large bone defects, cardiovascular diseases, osteogenesis imperfecta and orthopaedic injuries both in human and veterinary medicine. The clinical applications in which autologous MSCs can be used are limited because patient-specific tissue collection and cell expansion require time. For clinical applications in which MSCs should be used right away, it would be more practical to use cells collected from a donor, expanded in vitro and banked to be readily available when needed. However, there are concerns over the safety and the efficacy of allogeneic MSCs. The safety and efficacy of a therapy based on the use of allogeneic adipose tissue-derived mesenchymal stem cells (ASCs) associated to platelet rich plasma (PRP) were evaluated in 19 horses affected by acute or subacute overstrain superficial digital flexor tendonitis (SDFT). The application of allogeneic ASCs neither raised clinical sign of acute or chronic adverse tissue reactions, nor the formation of abnormal tissue in the long-term. After a follow-up of 24 months, 89.5% horses returned to their previous level of competition, while the reinjury rate was 10.5%, comparable to those recently reported for SDFT treated with autologous bone marrow derived MSCs. This study suggests that the association between allogeneic ASCs and PRP can be considered a safe and effective strategy for the treatment of SDF tendonitis in the horse.

Autologous bone marrow mesenchymal stromal cells for regeneration of injured equine ligaments and tendons: a clinical report.

The use of Mesenchymal Stromal Cells (MSCs) in orthopedic practice has recently and rapidly acquired an important role. Therapies based on the use of MSCs for the treatment of acute injuries as well as chronic inflammatory disorders are gradually becoming clinical routine. These cells have demonstrated intriguing therapeutic potentialities (i.e.: inflammation control, tissue regeneration and pathological scar prevention), that have been taken into consideration for use in both human and veterinary medicine. In particular, horses represent high performance athletes considered models for human pathologies since musculo-skeletal disorders frequently occur in this species. In the past, repair of tendon injures were performed by different methods. In particular, clinical therapy was based on ice application, bandage, box rest and controlled exercise. An alternative approach consisted on the use of corticosteroid (inflammation reduction) and other drugs (sodium hyaluronate, polysulphated glycosaminoglycans, beta aminoproprionitrile fumarate). Furthermore, surgical treatments like accessory ligament desmotomy, local irritation by line firing or pin firing were commonly used. More recently ultrasound, laser therapy, electromagnetic field therapy have been considered. Unfortunately, they did not allow complete tissue healing and quite often animals did not regain competitiveness. In order to minimize this inconvenience, the use of MSCs has been introduced as an alternative to the traditional approach since it represents a potential tool to improve tissue regeneration. Aim of this study was to evaluate the capability of MSCs to improve the functional outcome of horses affected by tendonitis and desmitis. Thirty-three breed and activity-matched horses affected by tendonitis or desmitis, were included in clinical trial scored for lesions and subdivided into two groups. Group 1 animals were treated with autologous MSCs, associated with platelet rich plasma (group 1). Bone marrow samples were collected from the sternum of the treated horses and processed in order to isolate MSCs. Following cell therapy, they were subjected to a rehabilitation period and their ability to resume training was evaluated. In this study, implanted MSCs caused no adverse reactions and thirteen out of the eighteen inoculated horses returned to race competitions. On the contrary, no improvement was seen in the twelve animals of group 2 treated with pin firing, that were not able to resume sport activity. In conclusion the clinical trial proves the safety of equine bone-marrow derived MSCs and a successful outcome of the treated animals that returned to their previous level of sport activity.

Risk of acquiring tick-borne infections in forestry workers from Lazio, Italy.

The seroprevalence of antibodies to Borrelia burgdorferi and tick-borne encephalitis (TBE) virus was evaluated in a group of forestry rangers in the Lazio region of Italy. One hundred and forty-five forestry rangers and 282 blood donors were examined by two-tiered serological tests for B. burgdorferi and TBE virus. Information on occupation, residence, tick bites, outdoor leisure activities and other risk factors was obtained. The prevalence of IgG/IgM antibodies to B. burgdorferi showed no statistical difference between the two groups, but there was a higher occurrence of IgM antibodies. There were significant differences between indoor and outdoor, urban and rural workplaces among the 145 exposed workers (χ² test: p < 0.001), and a higher risk for outdoor rural than urban tasks was detected among the ten Western blot-tested forestry rangers positive to B. burgdorferi (χ² test: p < 0.1). No seropositivity was observed for the TBE virus. Forestry rangers from the Lazio region did not have a higher risk of Borrelia infection than the blood donors, though an increase in the risk for outdoor tasks in a rural environment was observed.

Detection of different Borrelia burgdorferi genospecies in serum of people with different occupational risks: short report.

This study is aimed at applying a previously described PCR-based method to detect B. burgdorferi sensu lato and different Borrelia genospecies in total DNA preparations of serum samples collected from people with different occupational risks for tick bite and with serological evidence of borreliosis. Among the seropositive samples, the PCR for B. burgdorferi confirmed the positivity in 65 percent of the forestry workers and in 60 percent of the subjects living in the same area. None of the seronegative subjects belonging to the control group showed the presence of B. burgdorferi sensu lato DNA. Results on genospecies distribution show that B. afzelii was the predominant species, followed by B. garinii and finally by B. valaisiana.

[Emerging infectious diseases among swine workers]. / Patologie infettive emergenti per gli addetti ad allevamenti suinicoli.

In the last years emerging infections represent an important problem of public health and occupational medicine. Biological agents and their hosts exist in a precariously balanced and continuously evolving relationship, influenced by their environment. RNA viruses are responsible for most of the emerging diseases. Epidemics that recently affected the world of work are zoonoses, such as cases of SARS in healthcare staff Dutch poultry workers infected with the avian virus A/H7N7 in 2003, the current threat of avian flu A/H5N1 to poultry workers. Workers at risk include those who are in contact with live or dead infected animals, with aerosols, dust or surfaces contaminated by animal secretions, persons engaged in animal breeding and trade, veterinaries, and others. Pigs are at risk of acquiring many viral and bacterial diseases and, consequently, could be able to transmit some of these infections to occupationally exposed subjects. The aim of our study is to set out some emerging zoonosis that could affect swine workers, an occupational sector where a proper assessment of biological risks is difficult to perform.

[QuantiFERON-TB Gold test in homeless shelter staff: preliminary results]. / Test QuantiFERON-TB Gold in addetti all'assistenza: risultati preliminari.

Tuberculosis (TB) continues to be a major cause of disability and death and it has become a real problem in industrialized countries. The spread of HIV, the increasing immigration rate of people from countries with endemic TB and the growth of drug-resistant Mycobacterium tuberculosis strains extend its impact. Since the spread of the infection occurs early, health care workers are particularly exposed to the risk of contracting and/or transmitting the mycobacterium. In health care settings, policies and procedures for TB control should be developed, including health surveillance. Until recently the tuberculin skin test was the only available method for diagnosing tuberculosis, however it suffers several methodological weaknesses: high rate of false positive results in vaccinated populations, the subjectivity of the evaluation and the booster effect. Recently, the introduction of new in vitro serological tests, as the Quantiferon TB-Gold in tube (QFT-TB), may overcome these problems. The QFT-TB is based on the quantification of interferon-gamma released from sensitized lymphocytes in whole blood incubated overnight with PPD from M. tuberculosis and control antigens. The present study was performed on 27 nuns (homeless shelter staff) who were at risk for contracting tuberculosis. The prevalence of positive tests was 15 out 27 (55.5%).

[Biosafety in laboratories concerning exposure to biological agents]. / La biosicurezza nei laboratori per gli esposti ad agenti biologici.

Laboratory workers are exposed to a variety of potential occupational health hazards including those deriving from infectious materials and cultures, radiations, toxic and flammable chemicals, as well as mechanical and electrical hazard. Although all of them are significant, this paper will focus on biological hazards present in clinical and research laboratories. In fact, in spite of numerous publications, guidelines and regulations, laboratory workers are still subject to infections acquired in the course of their researches. This paper describes some aspects that include good microbiological practices (GMPs), appropriate containment equipment, practices and operational procedures to minimize workers' risk of injury or illness.

[SARS and occupational risk]. / SARS e rischio occupazionale.

SARS is an infectious disease caused by a previously unrecognized human coronavirus, called SARS-associated coronavirus (SARS-CoV). Current information indicates that most transmission is via respiratory droplets coming from a person who is symptomatic with SARS ("close contact"). The aim of our study is to evidence the critical role of the family physician, the first health-care worker who cares with suspected/probable SARS patients, underlying the importance of the correct use and management of the personal protective equipment.

Characterization of a candidate Trypanosoma brucei U1 small nuclear RNA gene.

We have previously shown that the poly(A) polymerase (PAP) gene of Trypanosoma brucei is interrupted by an intervening sequence. It was postulated that removing this intron by cis-splicing requires a yet unidentified U1 small nuclear RNA (snRNA), which in other organisms engages in base-pair interactions across the 5' splice site during early spliceosome assembly. Here we present a characterization of a 75 nucleotide long candidate T. brucei U1 snRNA. Immunoprecipitation studies indicate that a trimethylguanosine cap structure is present at the 5' end and that the RNA is bound to core proteins common to spliceosomal ribonucleoprotein particles. The U1 snRNA has the potential for extensive intermolecular base pairing with the PAP 5' splice site. We used block replacement mutagenesis to identify sequences necessary for in vivo expression of U1 snRNA. We found that at least two cis-acting elements, tRNA-like A and B boxes, located in the 5'-flanking region are necessary for U1 snRNA synthesis; no internal sequences close to the transcription start site are essential, suggesting a promoter architecture distinct from other trypanosome U-snRNA genes.