D. Di Luca

Human herpesvirus 6 infects the central nervous system of multiple sclerosis patients in the early stages of the disease

The presence and the replicative state of human herpesvirus 6 (HHV-6) were evaluated in clinical samples from multiple sclerosis (MS) patients at the first time of MS diagnosis. HHV-6 variant B was present in peripheral blood mononuclear cells of 5/32 (15%) patients, but persisted with a latent infection. Viral sequences were present also in cerebrospinal fluid (CSF), both free in the liquid (7/32, 22%) and latent in the cellular fraction (3/32, 9%), as shown by analysis of viral transcription. In these cases, variant A was detected. HHV-6 DNA sequences present in the CSF were associated to mature viral particles. In fact, in vitro infectious assays of CSF showed the presence of replication-competent virions. These results show that about 20% of MS patients have active foci of HHV-6 variant A infection in the early stages of the disease and suggest that viral replication takes place within the central nervous system.

B cell clonality in gastric lymphoid tissues of patients with Sjögren's syndrome.

OBJECTIVE: To determine the prevalence of mucosa associated lymphoid tissue (MALT) in the stomach and of a possible antigen driven proliferation, in patients with Sjögrens syndrome (SS). METHODS: Twenty one patients with primary SS and 80 dyspeptic controls underwent upper endoscopy. Lymphoid tissue and Helicobacter pylori were assessed by histopathological analysis. Epstein-Barr virus (EBV) or human herpes virus-6 (HHV-6) genome were studied by polymerase chain reaction (PCR) DNA amplification. Two PCR VDJ procedures were used to detect immunoglobulin heavy chain (IgH) gene rearrangement. RESULTS: Organised MALT was found in 33.3% of the patients, compared with 21.5% of the controls (NS). H pylori infection was seen in 71% of patients and 63% of controls. Genomic EBV or HHV-6 was found in a minor portion of SS gastric tissues. B cell expansion was detected in nine of the 21 patients. Infectious agents in the stomach might have contributed to B cell clonality only in 55.5% of the cases. No strict relationship was found between lymphoid follicles and clonality. CONCLUSION: Lymphoid accumulation in the gastric mucosa is common in Sjögrens syndrome, but full evidence for an antigen driven B cell expansion could not be demonstrated. Only a portion of those with clonal B cell expansion had evidence of an infectious agent. Other unknown infectious agents or factors related to the underlying disease (autoantigen) and its tissue environment may have a further role as possible causes of B clonal expansion in the gastric mucosa.

Chimeric BK Virus DNA Episomes in a Papillary Urothelial Bladder Carcinoma

BK virus (BKV) DNA sequences were identified in a papillary urothelial bladder carcinoma by Southern blot hybridization. The carcinoma contained both integrated and extrachromosomal DNA. Integrated sequences had a clonal restriction pattern, suggesting that BKV was integrated at some early stage of neoplastic initiation or progression. Viral episomes consisted of a population of covalent polymers based on a high-molecular-weight DNA unit, about 11-12 kb in size. DNA sequences non-homologous to the BKV genome were encompassed within DNA episomes, suggestive of acquisition of cellular sequences by viral DNA replication at the integration site. Extrachromosomal, chimeric DNA molecules were present at an average level of about 50 copies per cell, but their size, apparently incompatible with viral assembly, showed that BKV productive infection was impaired. The data suggest that infected cells underwent reversible changes affecting autonomous BKV DNA replication.

Panbacterial real-time PCR to evaluate bacterial burden in chronic wounds treated with Cutimed™ Sorbact™

The impact of polymicrobial bacterial infection on chronic wounds has been studied extensively, but standard bacteriological analysis is not always sensitive enough. Molecular approaches represent a promising alternative to the standard bacteriological analysis. This work aimed to assess the usefulness of a panbacterial quantitative real-time PCR reaction to quantitate the total bacterial load in chronic wounds treated with Cutimed™ Sorbact™, a novel therapeutic approach based on hydrophobic binding of bacteria to a membrane. The results obtained by panbacterial real-time PCR on conserved sequences of the bacterial 16S gene show that the bacterial burden significantly decreased in 10 out of 15 healing chronic wounds, and did not change in 5 out of 5 non-healing chronic wounds. On the contrary, classical culture for S. aureus and P. aeruginosa, and real-time PCR for Bacteroides and Fusobacterium did not show any correlation with the clinical outcome. Our study also shows that quantification of chronic wounds by panbacterial real-time PCR is to be performed on biopsies and not on swabs. These results show that panbacterial real-time PCR is a promising and quick method of determining the total bacterial load in chronic wounds, and suggest that it might be an important biomarker for the prognosis of chronic wounds under treatment.

Impact of sexual habits on the clinical evaluation of male HPV infection

A series of 199 male regular sexual partners of women attending an STD clinic for the examination and treatment of HPV-associated diseases was examined by peniscopy, surgical biopsy and nucleic acid hybridization for the presence of clinical, histological and molecular markers pathognomic of HPV infection. There was a 100% correlation between condylomata acuminata and detection of HPV type 6 or 11 DNA. Papillary lesions displayed neither histological signs of HPV infection, nor did they harbor HPV DNA (viral types 6, 11, 16, 18, 33) while 44.9% (22/49) of acetowhite epithelia showed HPV-suggestive histological changes. Of the 19 analysed for HPV DNA, 15.8% (3/19) harbored HPV 6/11 and 16 DNA. Regular male and female sexual partners did not always harbor the same HPV types, showing that latent or occult infection and the sexual habits of each individual play an important role in the clinical manifestations of HPV infection observed in sexual couples. The present data show that: i) the likelihood of developing a clinical HPV lesion was affected, to a large extent, by the previous sexual history and habits in the partners of women with flat condylomata, while partners of women with condylomata acuminata or CINs displayed a higher correlation with the current state of infection in their regular partner; ii) despite the assessed infective state of their consorts, men with a low lifetime number of sexual partners seldom displayed HPV-associated acetowhitening. Prevalence of such lesions, however, increased significantly with an increase in the total number of sexual partners; iii) clinical assessment and evaluation of HPV-risk for inconspicuous penile lesions in the male partner should be carried out not only on the basis of clinical and peniscopic appearance, but also considering the current state of infection in the regular partner and the sexual history and habits of each individual.

A study of some biologic properties of Bovid herpesvirus-4

This article summarizes the results of a study on several strains of Bovid herpesvirus-4 (BHV-4), isolated from cattle. The study had several objectives, namely, to verify (a) the disease-causing potential of the virus, (b) the possibility by BHV-4 to induce a latent infection in the natural host and (c) the entity of the relationships among strains of the virus isolated from different disease syndromes. The following data were obtained: (1) All strains tested were able to replicate in experimentally infected calves; however, only one strain (85/BH 16TV) caused an overt systemic disease. (2) The nervous system as well as the lymphoid structures appeared to be the target organs for replication of the virus. (3) BHV-4, like other herpesviruses, was able to establish latent infection in cattle. (4) When two strains of the virus, isolated from cattle affected by different disease syndromes, i.e. respiratory disease (strain DN-599) or vulvovaginitis (strain 85/BH 16TV), respectively, they resulted to be closely related to each other. In particular, they revealed a similar DNA pattern and both strains were able to cause respiratory disease in calves. Moreover, the two viral strains were mutually protective in that calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. (5) All BHV-4 strains tested generally failed to evoke a significant production of neutralizing antibody in the experimental calves.

Cerebrospinal fluid amounts of HLA-G in dimeric form are strongly associated to patients with MRI inactive multiple sclerosis

Background: The relevance of human leukocyte antigen (HLA)-G in dimeric form in multiple sclerosis (MS) is still unknown. Objective: To investigate the contribution of cerebrospinal fluid (CSF) HLA-G dimers in MS pathogenesis. Methods: CSF amounts of 78-kDa HLA-G dimers were measured by western blot analysis in 80 MS relapsing–remitting MS (RRMS) patients and in 81 inflammatory and 70 non-inflammatory controls. Results: CSF amounts of 78kDa HLA-G dimers were more frequent in RRMS than in inflammatory (p<0.01) and non-inflammatory controls (p<0.001) and in magnetic resonance imaging (MRI) inactive than in MRI active RRMS (p<0.00001). Conclusion: Our findings suggest that HLA-G dimers may be implicated in termination of inflammatory response occurring in MS.

Episomal HPV 16 DNA isolated from a cervical carcinoma presents a partial duplication of the early region

An invasive cervical carcinoma was found to harbor an episomal variant of human papillomavirus (HPV) type 16 DNA, with a size of about 10.1 kb. A genomic library of the tumor was constructed in bacteriophage lambda and a recombinant phage clone was isolated by screening with HPV 16 probe. Analysis by restriction mapping and Southern hybridization showed that the isolate contained a 2.2 kb duplication of the early region, which included part of E6, all E7 and part of E1 open reading frames. Possible consequences of this duplication for oncogenesis are discussed.

Comparative study of two strains of Bovid herpesvirus-4

Two strains of Bovid herpesvirus-4 (BHV-4), i.e. the prototype strain DN-599, obtained from a steer suffering of a respiratory disease, and the strain 85/BH 16TV, originated from a cow with vulvovaginitis, were compared in studies which included restriction endonuclease analysis, experimental infection and reciprocal cross protection tests. The restriction endonuclease analysis revealed that the resultant DNA patterns of the isolates were generally similar with only a difference in one fragment. The two strains were capable of causing respiratory tract infection in calves, even if they displayed a different level of virulence: the strain 85/BH 16TV being the most virulent while the strain DN-599 the least. The two viral strains were mutually protective in that the calves were generally found to be refractory to challenge inoculation with either the homologous or the heterologous virus. Finally, both viral strains failed to evoke the production of neutralizing antibody in the experimental calves.