Graves' orbitopathy, idiopathic orbital inflammatory pseudotumor and Epstein-Barr virus infection: a serological and molecular study.

OBJECTIVE: One of the hypotheses on the pathogenesis of autoimmune diseases, including Graves' disease (GD) and Graves' orbitopathy (GO), involves bacterial or viral infections. Recently, Epstein-Barr virus (EBV) has been proposed to play a role in the pathogenesis of idiopathic orbital inflammatory pseudotumor (IOIP) in Asians. The aim of the present study was to investigate the possible association of GO with EBV infection/exposure, as compared with IOIP, using serum and tissue samples, as well as primary cultures of orbital fibroblasts. METHODS: Thirty-one patients were studied, including four with IOIP, ten with GO, nine with GD without GO and eight control patients without IOIP, GD and GO. All patients with IOIP and GO underwent orbital decompression. Control patients underwent palpebral surgery. Fibroadipose orbital tissue samples were collected. Serum anti-EBV antibodies were measured in all patients. EBV-DNA was measured in blood samples, orbital tissue samples and primary cultures of orbital fibroblasts. RESULTS: Serum assays showed that the vast majority of patients have had a previous exposure to EBV, but no one had an acute infection. EBV-DNA was detected in ~40% of blood samples from GO, GD and control patients, but in none of the IOIP samples. EBV-DNA was not detected in any of the orbital tissue samples tested or in primary cultures of orbital fibroblasts. CONCLUSIONS: EBV infection does not seem to be associated with GD, GO and IOIP in Caucasians. Whether EBV is involved in IOIP in Asians or other populations remains to be confirmed.

Induced pluripotent stem cells in hematology: current and future applications.

Reprogramming somatic cells into induced pluripotent stem (iPS) cells is nowadays approaching effectiveness and clinical grade. Potential uses of this technology include predictive toxicology, drug screening, pathogenetic studies and transplantation. Here, we review the basis of current iPS cell technology and potential applications in hematology, ranging from disease modeling of congenital and acquired hemopathies to hematopoietic stem and other blood cell transplantation.

Absence of xenotropic murine leukemia virus-related virus in Italian patients affected by chronic fatigue syndrome, fibromyalgia, or rheumatoid arthritis.

The xenotropic murine leukemia virus-related virus (XMRV) has been recently linked to chronic fatigue syndrome in a US cohort in whom the virus was demonstrated in 67% patients vs 3.7% healthy controls. Albeit this finding was not substantiated by subsequent reports and eventually considered a laboratory contamination, the matter is still the object of intense debate and scrutiny in various cohorts of patients. In this work we examined well-clinically characterized Italian patients affected by chronic fatigue syndrome, and also fibromyalgia and rheumatoid arthritis, two chronic illnesses of basically unknown etiology which show quite a few symptoms in common with chronic fatigue syndrome. Although we used recently updated procedures and controls, the XMRV was not found in 65 patients with chronic fatigue syndrome diagnosis, 55 with fibromyalgia, 25 with rheumatoid arthritis, nor in 25 healthy controls. These results add to the ever-growing number of surveys reporting the absence of XMRV in chronic fatigue syndrome patients and suggest that the virus is also absent in fibromyalgia and rheumatoid arthritis.

Xenotropic murine leukaemia virus-related virus is not found in peripheral blood cells from treatment-naive human immunodeficiency virus-positive patients.

The human pathogen xenotropic murine leukaemia virus-related virus (XMRV) has been tentatively associated with prostate cancer and chronic fatigue syndrome. Unfortunately, subsequent studies failed to identify the virus in various clinical settings. To determine whether XMRV circulates in humans and the relationship with its host, we searched for the virus in 124 human immunodeficiency virus-infected patients who might have been exposed to XMRV, might be prone to infection as a result of progressive immunodeficiency, and had not yet been treated with antiretroviral drugs. Using nested PCR and single-step TaqMan real-time PCR, both designed on the XMRV gag gene, we could not find any positive samples. These findings add to the growing amount of scepticism regarding XMRV.

Immunosuppressive monoclonal antibodies: current and next generation.

Monoclonal antibodies (mAbs) are well-established therapeutics, as evidenced by the large number of Food and Drug Administration-approved mAbs for the treatment of cancers, and inflammatory or autoimmune diseases, and for the prevention and treatment of solid organ transplant rejection. Although, in many cases, mAbs have improved patient survival, they are also associated with an increased incidence of opportunistic infections. We review here the current and next generation of mAbs and the risks that infectious disease specialists should be aware of.

MMTV-like sequences in human breast cancer: a fluorescent PCR/laser microdissection approach.

The hypothesis that a retrovirus homologous to the mouse mammary tumour virus (MMTV) is involved in human breast cancer aetiology has fascinated scientists from many years, but it has never been convincingly demonstrated. Renewed interest in this hypothesis developed when an MMTV env gene-like sequence was found in 38% of human breast cancer tissues. Whereas some subsequent studies confirmed these findings, others did not. The main reasons for this discrepancy, among others, are the different sensitivities and technical details of current molecular approaches to the detection of these sequences. This study is an attempt to find sensitive and reproducible conditions capable of detecting MMTV env-like sequence in human samples. To this end, we first developed a fluorescence nested-PCR (FN-PCR) method that was able to detect very low copies of the viral genome, and then screened a panel of 45 frozen breast cancer samples obtained by laser microdissection. The MMTV env gene-like sequence was found in 15 (33%) of the human breast cancers analysed, whereas the same sequence was detectable neither in normal tissues nor in other types of tumour. Sequence analysis revealed 96% homology with the MMTV genome, but no other significant similarities with the human genome. The combined use of frozen material, microdissected cell populations and FN-PCR provides a novel, sensitive, robust, non-radioactive and fast methodology for the molecular detection of human-MTV. This approach might be successfully used in large molecular studies that aim to investigate the hypothesis of a retroviral aetiology of human breast cancer.

Changes in the SU can modulate the susceptibility of feline immunodeficiency virus to TM-derived entry inhibitors.

Feline immunodeficiency virus (FIV) provides a valuable animal model by which criteria for the development of HIV-1 inhibitors can be explored. Previous studies had shown that a synthetic 8-mer peptide modeled on the tryptophan-rich motif of the ectodomain of the viral transmembrane glycoprotein (TM) is a potent inhibitor of FIV The observation that inhibition efficiency varied somewhat depending on FV strain prompted the present study in which we investigated whether changes in the surface (SU) glycoprotein can affect virus susceptibility to TM-derived peptide inhibitors. This was done by examining how effectively selected entry inhibitors blocked the infectivity of well characterized variants and molecular clones of the prototype isolate of FIV The results have shown that substitutions in the SU can indeed modulate virus susceptibility to TM-derived entry inhibitors. Interestingly, we also observed a parallelism between reduced susceptibility to entry inhibitors and broad resistance to antibody-mediated neutralization.

Feline immunodeficiency virus-infected cat sera associated with the development of broad neutralization resistance in vivo drive similar reversions in vitro.

We previously reported that, upon reinoculation into cats, a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus constantly reverted to the broad neutralization resistance typical of primary virus isolates and identified residue 481 in the V4 region of the surface glycoprotein as a key determinant of the reversion. Here, we found that well-characterized immune sera, obtained from cats in which such reversion had occurred, selected in tissue culture in favor of virus variants that also had a neutralization-resistant phenotype and had amino acid 481 changed, thus indicating that the host's humoral immune response is capable of driving the reversion in the absence of other intervening factors. In contrast, a second group of immune sera, elicited by a virus variant that had already reverted to neutralization resistance in independent cats, induced the emergence of escape mutants lacking broad neutralization resistance and neutralized fewer virus variants. It is proposed that the viral variants used to produce the two sets of sera may have generated different antibody repertoires.

During readaptation in vivo, a tissue culture-adapted strain of feline immunodeficiency virus reverts to broad neutralization resistance at different times in individual hosts but through changes at the same position of the surface glycoprotein.

The broad resistance to antibody-mediated neutralization of lentiviruses recently isolated from infected hosts is a poorly understood feature which might contribute to the ability of these viruses to persist and to the failure of experimental vaccines to protect against virulent viruses. We studied the underlying molecular mechanisms by examining the evolution of a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus upon reinoculation into specific-pathogen-free cats. Reversion to broad neutralization resistance was observed in seven of seven inoculated animals and, in individual hosts, started to develop between less than 4 and more than 15 months from infection. After comparison of the envelope sequences of the inoculum virus, of an additional 4 neutralization-sensitive in vitro variants, and of 14 ex vivo-derived variants (6 neutralization sensitive, 5 resistant, and 3 with intermediate phenotype), a Lys-->Asn or -->Glu change at position 481 in the V4 region of the surface glycoprotein appeared as a key player in the reversion. This conclusion was confirmed by mutagenesis of molecularly cloned virus. Analysis of viral quasispecies and biological clones showed that the intermediate phenotype was due to transient coexistence of neutralization-sensitive and -resistant variants. Since the amino acid position involved was the same in four of four recent revertants, it is suggested that the number of residues that control reversion to broad neutralization resistance in FIV might be very limited. Amino acid 481 was found to be changed only in one of three putative long-term revertants. These variants shared a Ser-->Asn change at position 557 in region V5, which probably collaborated with other mutations in long-term maintenance of neutralization resistance, as suggested by the study of mutagenized virus.

High prevalence of TT virus (TTV) and TTV-like minivirus in cervical swabs.

Genomes of TT virus (TTV) and TTV-like minivirus DNA were detected in 80% and 61% of cervical swabs from healthy women, respectively, regardless of concurrent human papillomavirus infection. These results show that the potential exists for sexual transmission of TTV and related viruses.

Tumor necrosis factor-alpha and virus expression in the central nervous system of cats infected with feline immunodeficiency virus.

Cytokine disregulation has been implicated in the pathogenesis of lentivirus-induced diseases. In the present study, 18 specific pathogen free (SPF) cats were inoculated with feline immunodeficiency virus (FIV) Petaluma strain and sacrificed at different times post-infection. Five additional SPF cats were used as controls. The cell localization of the cytokine tumor necrosis factor alpha (TNF-alpha) in the central nervous system (CNS) was determined by immunohistochemical and morphometric analyses with a polyclonal rabbit anti-human TNF-alpha antibody. TNF-alpha and FIV RNA were measured using competitive reverse transcriptase polymerase chain reaction (PCR) assays and the number of proviral genomes was estimated by competitive PCR. Portions of frontal cortex were collected from each animal and both formalin-fixed and snap-frozen and stored at -80 degrees C until used. The results showed that TNF-alpha is present mainly in astrocytes and microglial cells. Morphometric analysis showed that areas of TNF-alpha production increased in the early phases of infection. Molecular analyses demonstrated that the kinetics of proviral loads in the CNS were comparable to what observed in lymph nodes and peripheral blood mononuclear cells, with the peaks in the early and late stages of infection. A positive correlation was found between viral parameters and TNF-alpha transcription, the strongest relationship was found between the transcription of the cytokine and viral RNA load. These results confirm that invasion of CNS by FIV occurs soon after virus exposure and that during this phase there is an increase of local viral loads with concomitant up-regulation of TNF-alpha expression. During the asymptomatic phase viral replication remains low in spite of the progression of CNS alterations. The dissociation between the viral load and the lesions observed suggests the importance of an indirect mechanism for the progression of these lesions, even if TNF-alpha seems to play a role particularly in the early phase of infection.

Antiviral properties of a crude extract from a green alga Caulerpa taxifolia (Vahl) C. Agardh.

A crude extract of Caulerpa taxifolia was tested for its antiviral activity. The chloroform-methanol residue showed an interesting inhibitor effect in vitro toward the feline immunodeficiency virus (FIV), a valid model for studying the acquired immunodeficiency syndrome. This extract reduced the virus-induced syncytia in the cultured cells, the viral reverse transcriptase activity and the viral capsid protein P24 expression.

Susceptibility of human and non-human cell lines to HCV infection as determined by the centrifugation-facilitated method.

The centrifugation-facilitated inoculation method was used to test 51 human and non-human cell lines for ability to support HCV replication. As determined by nested RT-PCR, one fifth of the cell lines tested were virus positive 15 days post inoculation suggesting that the centrifugation-facilitated inoculation is an efficient method for cell infection with HCV. However, virus production by infected cultures remained of low grade, thus showing that the unknown factors which limit HCV replication in vitro are not overcome by the procedure.

Detection of feline immunodeficiency proviral sequences in lymphoid tissues and the central nervous system by in situ gene amplification.

The availability of sensitive methods for detecting and localising the feline immunodeficiency virus (FIV) may help shed light on its role in generating tissue damage observed during infection. As immunohistochemical and in situ hybridisation techniques might not be sufficiently sensitive for this type of study, we adapted to FIV PCR-in situ hybridisation (PCR-ISH) that combine the extreme sensitivity of PCR with the precise localisation provided by ISH. The steps important for the success of PCR-ISH, such as sample preparation, permeabilisation, amplification profile, type of labels, and hybridisation conditions were optimised using paraformaldehyde-fixed and formalin-fixed paraffin-embedded sections of cells infected in vitro with FIV. As controls for amplification, the feline tumor necrosis factor-alpha gene (TNF-alpha) and the non-related EBNA-1 gene of the human Epstein-Barr virus were used. Once the method proved sufficiently sensitive and specific with these cells, the PCR-ISH assay was applied to paraffin sections of the lymph nodes, spleen and central nervous system of a 2-year FIV infected cat that, at the time of challenge, harboured low copy numbers of proviral genomes. Comparison of the results of PCR-ISH, competitive PCR and immunohistochemical analysis are described.

AIDS vaccination studies using an ex vivo feline immunodeficiency virus model: homologous erythrocytes as a delivery system for preferential immunization with putative protective antigens.

Feline immunodeficiency virus (FIV) is a useful model for testing of criteria for AIDS vaccine development. In the protocol we adopted, we used a primary isolate of FIV as a source of antigen and, for challenge, plasma from cats infected with the homologous virus never passaged in vitro. Cat erythrocytes (RBC) were coated with the surface components of freshly harvested and purified FIV by means of biotin-avidin-biotin bridges and used to immunize specific-pathogen-free cats (four doses at monthly intervals; total amount of FIV antigen administered per cat, approximately 14 microg). Immunized cats developed moderate levels of antibodies directed mainly to surface components of the virion and clearly evident lymphoproliferative responses. Four months after the last dose of immunogen, FIV-immunized cats and control cats immunized with bovine serum albumin-coated RBC were challenged. Judged from the results of the subsequent 12-month follow-up, FIV-immunized cats exhibited at least some degree of protection. However, following rechallenge, most of the FIV-immunized animals became virus positive in spite of a booster immunogen dose given 2 months before the second challenge.

Differences in hepatitis C virus quasispecies composition between liver, peripheral blood mononuclear cells and plasma.

Hepatitis C virus (HCV) exists in vivo as a highly variable mixture of closely related genomes (quasispecies), but the pathogenetic significance of such heterogeneity is still largely unknown. To investigate this issue, we compared the composition of HCV quasispecies found in the liver, peripheral blood mononuclear cells (PBMC) and plasma of ten patients by single-strand conformation polymorphism analysis of the E2/NS1 region and sequencing of the variants detected. We found considerable quasispecies differences between the liver and PBMC in all the patients, involving variant numbers, relative quantities and relative electrophoretic mobilities, but no apparent tissue-specific trend. Genome variants present in the liver and/or PBMC were not detected in the corresponding plasma samples, while certain HCV variants were present only in plasma. No dominant amino acids or amino acid pattern characteristic of variants present solely in the PBMC were detected in the E2/NS1 region sequenced.

Renal involvement in feline immunodeficiency virus infection: p24 antigen detection, virus isolation and PCR analysis.

Renal alterations characterized morphologically by glomerular and tubulo-interstitial lesions and clinically by a heavy proteinuria and sometimes by renal failure are frequent in feline immunodeficiency virus (FIV) infected cats. To investigate the possible role of local FIV replication in the genesis of this renal damage, renal tissues of 15 consecutive naturally infected and five non-infected cats were examined for traces of the virus by immunohistochemistry, using a monoclonal anti-p24 antibody in a streptavidin-biotin peroxidase labeled system, cultivation and polymerase chain reaction (PCR). Tubular epithelial cells as well as scattered interstitial inflammatory and glomerular cells were positive for p24 antigen in 13 cats. Viral isolation was successful in seven cats, and FIV gag DNA and RNA sequences were detected in 14 and five cats, respectively. Control cats were constantly negative. Although not conclusive, these results suggest that a direct role of FIV in the induction of the renal damage observed in infected animals is possible.