Enzymatic escharolysis with nexobrid® on partial thickness burn wounds: pre- and post-debridement histological assessment.

Enzymatic escharolysis is an innovative, non-surgical treatment method for severe burn patients as it allows very early, nontraumatic removal of necrotic tissue even on patients whose overall clinical conditions would mandate delaying traditional surgical escharectomy. The aim of this work was to examine aspects related to the "quality" of enzymatic debridement, which is inherently different from surgical debridement. To this end, biopsies harvested from partial thickness burn wounds, before and after enzymatic treatment, were histologically assessed. As is well known, surgical escharectomy removes the necrosis as well as some of its neighbouring healthy tissue, sharply and radically, leaving a perfectly clean and viable wound bed. On the other hand, enzymatic escharolysis is more selective, as it completely wipes out the necrotic portion while sparing unharmed and partially damaged tissue. In this study, only mid-deep partial thickness wounds were examined, and it was observed that partially damaged dermis was always spared by the lytic action. This dermis, however, showed some "homogenization" characteristics, had few vital skin annexes in it, and therefore looked very similar to the scaffold of dermal matrices currently available on the market. This scaffold should be safeguarded with a view to possibly achieving a more complete and functional spontaneous tissue regeneration. Conversely, if this dermal portion is mismanaged, it could desiccate, thus leading to the formation of a neo-eschar with unpredictable clinical evolution. Understanding how escharolysis actually works allowed us to extrapolate fruitful usage suggestions to optimize the procedure and fully exploit its potential.

La détersion enzymatique est une technique innovante non chirurgicale permettant l'ablation très précoce et non traumatique des tissus nécrosés même chez des patients dont l'état général nécessiterait de repousser une excision chirurgicale. Le but de ce travail était d'évaluer la « qualité ¼ du débridement enzymatique, par essence différent du traitement chirurgical. À cette fin, nous avons examiné histologiquement des biopsies réalisées avant et après détersion. Il est bien connu que la chirurgie emporte totalement et radicalement la nécrose et une partie du tissu environnant, laissant en place un tissu parfaitement propre et viable. Le débridement enzymatique est plus sélectif, emportant tout le tissu nécrosé sans affecter les tissus sains ou viables. Cette étude ne s'est intéressée qu'aux brûlures intermédiaires et nous avons observé que les régions saines étaient toujours préservées. Ce derme restant apparaît toutefois homogénéisé, avec peu d'annexes viables ce qui fait penser aux matrices des dermes artificiels actuellement commercialisés. Il doit être préservé afin de promouvoir une régénération tissulaire complète et fonctionnelle. Ainsi, si ce derme restant n'est pas correctement pris en charge, il peut se dessécher et aboutir à la formation d'un nouvel escarre, d'évolution imprévisible. Le compréhension du mécanisme exact de la lyse de la brûlure permet de développer des protocoles d'optimisation de la technique de lyse enzymatique.

VIP restores natural killer cell activity depressed by hepatitis B surface antigen.

Vasoactive intestinal peptide (VIP) has recently been shown to bind to human lymphocytes and modulate immune functions. The ability of VIP in restoring natural killer (NK) cell activity depressed by hepatitis B surface antigen (HBsAg) has been investigated in the present research. Human lymphocytes were incubated with HBsAg and, after washing, a 4-hr cytotoxicity assay was performed. VIP was coincubated with lymphocytes during the preincubation with HBsAg or, alternatively, throughout the cytotoxicity assay. The study revealed that VIP, either preincubated or coincubated in the 4-hr assay, strongly restores NK cell activity depressed by viral antigen. This is noteworthy considering that a number of lymphocyte modulators such as interferons fail in restoring viral-dependent NK cell activity depression. In contrast with previous reports, even when coincubated in the 4-hr assay, VIP is a strong activator of NK cell activity. Further studies will be required to understand which mechanisms are involved in the interrelation between VIP and NK cells during viral infections.

Human polyomavirus BK (BKV) load and haemorrhagic cystitis in bone marrow transplantation patients.

Several observations suggest an association between long-lasting haemorrhagic cystitis (HC) in bone marrow transplantation (BMT) recipients and human polyomavirus BK (BKV) reactivation, but no conclusive evidence has been obtained so far. The amount of BKV measured in the urine of BMT patients during an episode of HC was compared with that detected in the urine of BMT patients without HC and of immunocompetent individuals in order to better assess the association of BKV reactivation with HC. For this purpose a quantitative competitive PCR was developed. The application of this assay to clinical samples allowed us to distinguish asymptomatic reactivation both in healthy individuals and in immunocompromised patients from reactivation associated with HC, in almost all cases. Low levels, below the sensitivity of the quantitative assay, were shown in asymptomatic healthy individuals and in about 50% of immunocompromised patients. A significantly higher viral load than in the urine of asymptomatic immunocompromised patients was detected in the urine of patients with HC. These data strengthen the hypothesis that BKV reactivation can cause, together with other factors, the majority of late HC in BMT recipients as well as in patients treated for acute refractory lymphoblastic leukemia.

Monitoring of polyomavirus BK viruria in bone marrow transplantation patients by DNA hybridization assay and by polymerase chain reaction: an approach to assess the relationship between BK viruria and hemorrhagic cystitis.

An association between long-lasting hemorrhagic cystitis (HC) in bone marrow transplantation (BMT) patients and viral infections, mostly with reactivation of the human polyomavirus BK (BKV), is suggested by several previous reports. We have carried out a prospective study in 55 (30 allogeneic, 24 autologous, 1 syngeneic) BMT recipients with the aim of evaluating the role of BKV in this frequent complication after BMT. To overcome the well known difficulties in BK virus isolation in cell cultures, a DNA hybridization assay and the polymerase chain reaction (PCR) were used for the detection and monitoring of viral urinary shedding, respectively. The presence of human polyomavirus JC and adenovirus DNA was also sought in urine specimens. BK viruria was demonstrated in 52.7% of patients (in 53.3% allogeneic and in 54.1% autologous BMT), whereas JCV and adenovirus were seldom present. Seven cases of HC (20% in allogeneic and 4% in autologous BMT) occurred and in all cases the clinical event was associated with BKV urinary shedding. This study suggests that BKV infection alone does not invariably lead to HC in BMT patients; for HC to occur the presence of other, at present unidentified, factors seems to be necessary.

Polymerase chain reaction for synthesis of digoxigenin-labelled DNA probe: application to parvovirus B19 and to polyomavirus BK.

A one-step polymerase chain reaction (PCR) was used to synthesize a digoxigenin-labelled probe, 176 bp long, for the detection of human polyomavirus BK (BKV). A 104 bp-long digoxigenin-labelled probe was generated by 'nested' PCR for the detection of human parvovirus B19 (virus B19). In both cases the whole viral genome was used as template. Different amounts of template as well as different percentages of dTTP substituted by digoxigenin-dUTP (dig-dUTP) in the reaction mixture were employed in order to determine the optimum conditions for the labelled probe synthesis. The sensitivity and the specificity of these PCR-produced probes, together with the simplicity and the reduced time scale of the procedure, suggest the potential of this technique as an additional method for preparing non-radioactive molecular probes for routine diagnosis of viral infections.

Isolation of influenza A(H3N2) virus with "O"-->"D" phase variation.

We report the isolation of two influenza A(H3N2) virus strains which were unable, in the first passages in MDCK cell culture, to agglutinate chicken erythrocytes, though reacting with guinea pig and turkey red blood cells. This observation demonstrates that the occurrence of this phenomenon is not exclusive to influenza A(H1N1) viruses, as previously reported. In order to investigate the molecular basis of this phenomenon, we analysed the nucleotide sequence of the HA-1 region, presumed to be involved in the switch of haemagglutination properties, from the virus present in the original samples and in the corresponding strains isolated and cultivated in MDCK cells and in embryonated eggs. The substitution of amino acid 138 (Ala-->Ser) in MDCK cells could be related to the change in haemagglutination characteristics.

Human herpesvirus 8 DNA sequences are present in bone marrow from HIV-negative patients with lymphoproliferative disorders and from healthy donors.

Bone marrow (BM) from patients affected by multiple myeloma (MM), exhibiting monoclonal gammopathy of undetermined significance (MGUS) or with non-Hodgkin lymphoma (NHL) as well as from healthy donors were investigated for the presence of human herpesvirus-8 (HHV-8) DNA sequences. ORF 26 sequences were detected in 36--56% of the patients and in 29% of the controls. In a few cases, two other HHV-8 DNA sequences were also detected. These observations indicate that the presence of the HHV-8 genome in BM is relatively common in different groups of patients as well as in healthy individuals and do not support an alleged role for HHV-8 in MM.

Human parvovirus B19 infection in bone marrow transplantation patients.

We report the results of a survey of parvovirus B19 infection carried out with the aim to evaluate the frequency and the role of this infection in bone marrow transplant (BMT) recipients, as it is known that B19 virus can persist in clinical circumstances of immunodeficiency. Fifty-one patients subjected to BMT in the Bone Marrow Transplantation Center of Florence were enrolled in this study. Immunological and virological indications of B19 infection were tested weekly during the stay in hospital. A high rate of seroconversion or B19 antibody rise was observed, but, in absence of B19 IgM or B19 DNA presence, this result seems to be attributable to a passive immunization, rather than to a recent viral infection. In these 51 patients, as well as in 59 others not included in this study, clinical manifestations imputable to B19 infection have never been observed. It is possible that the isolation measures and the intravenous immunoglobulins (IVIG) administration may contribute in preventing B19 infection in the BMT recipients at least until the hospital discharge.

Human parvovirus B19 infection in hemophiliacs first infused with two high-purity, virally attenuated factor VIII concentrates.

Human parvovirus B19 can be transmitted by coagulation factor concentrates and is highly resistant to virucidal methods. To evaluate whether the additional removal of virus by chromatographic methods during the manufacture of high-purity concentrates reduces the risk of B19 transmission, we have prospectively evaluated the rate of anti-B19 seroconversion in two groups of susceptible (anti-B19 negative) hemophiliacs infused with high-purity, heated (pasteurized) or solvent-detergent-treated factor VIII concentrates. Both products infected a relatively high proportion of patients (nine of 20).

Archetypal and rearranged sequences of human polyomavirus JC transcription control region in peripheral blood leukocytes and in cerebrospinal fluid.

Two forms of human polyomavirus JC (JCV) genome are known based upon the structure of the transcriptional control region (TCR) of the virus: the archetypal form, which is commonly detected in urine, and the rearranged form, which was first detected in brain tissue from progressive multifocal leukoencephalopathy (PML) patients. The latter actually includes a group of TCR variants that, relative to the former, are characterized by various deletions and/or duplications. The aim of this study was to establish whether or not a correlation exists among the TCR type, the spreading of the virus within the host and its ability to cause PML. JCV TCR sequences from peripheral blood leukocytes (PBL) and cerebrospinal fluid (CSF) obtained from various groups of patients were compared. JCV with archetypal TCR was detected in CSF and PBL specimens from patients without neurological disorders or who eventually received a diagnosis of a non-PML neurological disorder. Rearranged TCR sequences were detected in all the CSF and PBL specimens from PML patients. The high similarity observed between the TCR structure detected in PBL and CSF specimens from individual patients could strengthen the hypothesis that PBL has a role in spreading JCV to the brain. Moreover, heterogeneous TCR patterns have been shown in individual PBL specimens from PML patients. This supports the hypothesis that, in PBL, JCV may replicate and undergo rearrangements of the TCR. The detection of JCV DNA by PCR in CSF independently from PML, although rare, could suggest that this assay is not sufficient for a virological diagnosis of PML. Further studies are required to assess the usefulness of quantitative assays or TCR typing in combination with PCR for diagnostic purposes.

Human polyomaviruses DNA detection in peripheral blood leukocytes from immunocompetent and immunocompromised individuals.

Peripheral blood leukocytes from immunocompetent and immunocompromised individuals were analyzed for human polyomarivus BK and JC DNA presence. A nested polymerase chain reaction which amplify the transcriptional control region of the genome of both viruses was employed. The immunocompromised patients included bone marrow transplantation recipients and AIDS patients. BKV sequences were detectable in 52.8-62.5% of the individuals included in this study, whereas the percentage of individuals with JCV sequences in peripheral blood lymphocytes varied from 38.8% to 50%. The frequency of reactivations of BKV and JCV were also determined by detection of shedding in urine of viral DNA. The highest frequency of reactivations of either BKV or JCV was demonstrable in the group of bone marrow transplantation recipients, but reactivations occurred also in immunocompetent individuals. JCV sequences amplified from urine samples showed a restriction pattern similar to the archetype one, whereas sequences obtained from lymphocytes showed rearranged pattern as well as archetype pattern. Finally all JCV sequences from cerebrospinal fluid seemed to be rearranged. These observations suggest that peripheral blood lymphocytes have a fundamental role in the persistence of polyomaviruses infection and in the dissemination at least of JCV within the organism allowing that rearranged variants, better adapted to grow in brain tissue, emerge.