A new contrast agent for radiological and dissection studies of the arterial network of anatomic specimens.

PURPOSE: The aim of the present study is to propose a new contrast agent that can be easily applied both to CT and dissection studies to replace lead oxide based formulas for comparative anatomical analyses of the vascularisation of cadaveric specimens. METHODS: The infusion material was an epoxy resin, especially modified by the addition of barium sulphate to enhance its radiopacity. The final copolymer was toxicologically safe. To test the properties of the new material, several cadaveric limb injections were performed. The injected specimens were both CT scanned to perform 3D vascular reconstructions and dissected by anatomical planes. RESULTS: There was a perfect correspondence between the image studies and the dissections: even the smallest arteries on CT scan can be identified on the specimen and vice versa. The properties of the epoxy allowed an easy dissection of the vessels. CONCLUSIONS: The new imaging techniques available today, such as CT scan, can evaluate the vascular anatomy in high detail and 3D. This new contrast agent may help realising detailed vascular studies comparing CT scan results with anatomical dissections. Moreover, it may be useful for teaching surgical skills in the field of plastic surgery.

Single-nucleotide polymorphism-defined class I and class III major histocompatibility complex genetic subregions contribute to natural long-term nonprogression in HIV infection.

We performed a genome-wide association study comparing a cohort of 144 human immunodeficiency virus (HIV type 1-infected, untreated white long-term nonprogressors (LTNPs) with a cohort of 605 HIV-1-infected white seroconverters. Forty-seven single-nucleotide polymorphisms (SNPs), located from class I to class III major histocompatibility complex (MHC) subregions, show statistical association (false discovery rate, <0.05) with the LTNP condition, among which 5 reached genome-wide significance after Bonferonni correction. The MHC LTNP-associated SNPs are ordered in ≥4 linkage disequilibrium blocks; interestingly, an MHC class III linkage disequilibrium block (defined by the rs9368699 SNP) seems specific to the LTNP phenotype.

Wild ungulate slaughtering and meat inspection.

The different phases of production of farmed and hunted wild game fresh meat are described. The importance of reducing the stress resulting from handling procedures (capture, restraint, transport) before the slaughtering of animals is highlighted, due to its adverse effects on meat quality. The hygienic and animal welfare criteria to be adopted in the slaughtering of wild game are described. The importance of carcass inspection immediately after slaughtering is stated, so that meat can be destined for human consumption. Possible alterations occurring in fresh and refrigerated meat, that are capable of compromising its consumability, are presented.

Effect of udder health status and lactation phase on the characteristics of Sardinian ewe milk.

Mammary involution and inflammation are known to negatively affect milk quality. A trial was carried out to elucidate the mechanism by which udder health status and lactational phase determine compositional modifications in ovine milk. A total of 60 individual milk samples was collected from a group of 20 pluriparous Sardinian ewes from mid to late lactation. Each sample was assessed for its chemical characteristics, quantitative distribution of casein fractions, lactodynamographic characteristics, and enzymatic activity. Udders were classed as healthy, doubtful, or infected on the basis of repeated somatic cell counts, and samples were grouped in 3 classes of days in milk. Results indicated that both udder inflammation and mammary involution can increase plasmin (PL) activity (15.6 vs. 18.4 U/mL in healthy vs. infected udders; 14.0 vs. 20.2 U/mL in phase 1 vs. 3), which is responsible for an evident protein breakdown in milk. Significant differences between groups were observed for several characteristics. With regard to udder heath status, casein index was lower in the infected vs. healthy udders (74.8 vs. 76.6%), and beta(tot)-casein showed a similar trend (43.9 vs. 46.6%). As a consequence of protein degradation, gamma-casein (5.78 vs. 2.82%) and proteolysis index (7.60 vs. 3.82) increased in the infected group with respect to the healthy group. Udder health status also affected milk technological traits. Udder inflammation resulted in longer clotting time (20.7 vs. 16.5 min for infected vs. healthy, respectively) and in poorer curd firmness (35.6 vs. 47.6 mm for infected vs. healthy, respectively). Frequency of samples reactive to rennet was 100, 93, and 67%, respectively, for healthy, doubtful, and infected groups. With regard to lactational phase, a decrease in alpha(s1)-casein (39.13 vs. 29.36%) and beta(1)-casein (23.41 vs. 19.36%) occurred during phase 1 vs. 3, whereas kappa + alpha(s2)-casein increased (12.30 vs. 21.56%, phase 1 vs. 3). Correlation coefficients confirmed the role of PL in protein degradation. It was concluded that PL activity was strongly affected by both lactational phase and udder health status and, in turn, could be an important agent enhancing milk quality detriment.

The MHC class II transactivator: prey and hunter in infectious diseases.

The MHC class II transcriptional activator (CIITA) is the major regulator of expression of MHC class II genes. Thus, CIITA plays a fundamental role in the regulation of the immune response. Here, we discuss our findings on the dual role of CIITA during infections, as the target (prey) for certain pathogens but the host effector (hunter) against other pathogens, including HIV-1. This dual role is placed in an evolutionary context as a rather peculiar example of a strategy used by pathogens to evade host defenses and a counteraction of the host to minimize the survival and spread of the pathogen.

Human T cell leukemia virus type II increases telomerase activity in uninfected CD34+ hematopoietic progenitor cells.

The aging process of long-term self-renewing hematopoietic stem/progenitor cells is not yet completely understood and recent studies on antiapoptotic cell pathways have demonstrated a close linkage between telomerase activation and Bcl-2 deregulation in human cancer cells. The present work shows that human T cell leukemia virus type II (HTLV-II) Mo virions that have originated from the T cell line (C344), but not from the B cell line (BJAB), are critically involved in mediating survival and growth effects on hematopoietic precursors (represented by both the TF-1 CD34+ cell line and by peripheral blood-derived CD34+ cells) through the maintenance or enhancement of telomerase activity and the induction of bcl-2 expression. In addition, using an interleukin-3-dependent TF-1 cell line, it was demonstrated that IL-3 deprivation was sufficient to influence the levels of telomerase activity and Bcl-2 expression in CD34+ cells. Taken together, these findings suggest that, in appropriate conditions, extended hematopoietic progenitor cell survival and proliferation following HTLV-II exposure depends on a synergistic interaction between up-regulation of Bcl-2 and activation of telomerase activity.

HTLV-II down-regulates HIV-1 replication in IL-2-stimulated primary PBMC of coinfected individuals through expression of MIP-1alpha.

The influence of human T-cell leukemia/lymphoma virus type II (HTLV-II) in individuals also infected with HIV-1 is poorly understood. To evaluate the reciprocal influence of HTLV-II and HIV-1 infection, primary peripheral blood mononuclear cell (PBMC) cultures from coinfected individuals were established in the presence of interleukin 2 (IL-2). In these cultures, the kinetics of HTLV-II replication always preceded those of HIV-1. Noteworthy, the kinetics of HIV-1 production were inversely correlated to the HTLV-II proviral load in vivo and its replication ex vivo. These observations suggested a potential interaction between the 2 retroviruses. In this regard, the levels of IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) were measured in the same coinfected PBMC cultures. Endogenous IL-2 was not produced, whereas IL-6 and TNF-alpha were secreted at levels compatible with their known ability to up-regulate HIV-1 expression. The HIV-suppressive CC-chemokines RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta were also determined in IL-2-stimulated PBMC cultures. Of interest, their kinetics and concentrations were inversely related to those of HIV-1 replication. Experiments were performed in which CD8(+) T cells or PBMCs from HTLV-II monoinfected individuals were cocultivated with CD4(+) T cells from HIV-1 monoinfected individuals separated by a semipermeable membrane in the presence or absence of antichemokine neutralizing antibodies. The results indicate that HTLV-II can interfere with the replicative potential of HIV-1 by up-regulating viral suppressive CC-chemokines and, in particular, MIP-1alpha. This study is the first report indicating that HTLV-II can influence HIV replication, at least in vitro, via up-regulation of HIV-suppressive chemokines. (Blood. 2000;95:2760-2769)

The origin and evolution of human T-cell lymphotropic virus type II (HTLV-II) and the relationship with its replication strategy.

In this review, the origin and evolution of the human T-cell lymphotropic virus type II (HTLV-II) are discussed, with particular emphasis on its high genomic stability. In particular, it appears that the virus originated in the African continent and has been infecting human populations for several thousands of years. The very low divergence accumulated on average between different viral strains during such a long period could be explained by considering that in infected individuals the viral amplification could be due mainly to the clonal expansion of the infected cells, via cellular mitosis, rather than to reverse transcription. HTLV-II was introduced into the American continent during one or more migrations of HTLV-II-infected Asian populations over the Bering land bridge, some 15,000-35,000 years ago. Finally, during the last few decades, HTLV-II has been transmitted from native Amerindians to injecting drug users (IDUs). It might be speculated that at least two separate introductions of HTLV-II in European IDUs from US IDUs have occurred, due to the practice of needle-sharing among IDUs.

Evolutionary rate and genetic heterogeneity of human T-cell lymphotropic virus type II (HTLV-II) using isolates from European injecting drug users.

Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 x 10(-4) and 2.7 x 10(-5) nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10(-2) and 10(-4).

Human T-cell leukemia virus type II directly acts on CD34+ hematopoietic precursors by increasing their survival potential. envelope-associated HLA class II molecules reverse this effect.

The role of human T-cell leukemia virus type II (HTLV-II) in human lymphoproliferative and hematopoietic abnormalities in which the retrovirus can be isolated is still elusive. Here we show that the C344 T-cell-derived lymphotropic HTLV-II type IIa Mo strain acts directly on CD34+ hematopoietic precursors by rescuing them from apoptosis induced by interleukin-3 (IL-3) deprivation. This effect is viral strain-specific, as it is not observed with the B-lymphotropic HTLV-II type IIb Gu strain, it does not require infection of the hematopoietic precursors, and, interestingly, it is strongly dependent on the infected cellular host from which the virus was derived. Indeed, growth adaptation of the Mo strain to the permissive B-cell line, BJAB, renders the virus no longer capable of mediating the antiapoptotic effect. However, pretreatment of the BJAB-adapted Mo strain with antibodies specific for HLA class II, but not class I, histocompatibility antigens restores the antiapoptotic potential of the virus. These results constitute the first evidence that HTLV-II retrovirus can directly influence the homeostasis of human progenitors, without infecting them, and that this crucial activity is strongly inhibited by the presence of host-derived envelope-associated HLA class II antigens.

Clonal expansion of human T-cell leukemia virus type II in patients with high proviral load.

In previous studies we demonstrated that individuals infected by human T-cell leukemia virus type II (HTLV-II) presented a high degree of variation in proviral load and that the cellular tropism of this virus was expanded in some patients to B-lymphocytes. To understand whether the observed high proviral load could be associated with the clonal expansion of the infected cells, we have studied the mode of integration of HTLV-II in six infected individuals with proviral load higher than 1% of total peripheral blood mononuclear cells (PBMCs). An inverse polymerase chain reaction (PCR) analysis, which allowed the amplification of the region flanking the 5' end of the provirus, was developed for HTLV-II. A single band, corresponding to a monoclonal expansion, was found in four of six patients analyzed, while in the other two patients an oligoclonal type of integration was observed. The results for inverse PCR analysis were confirmed by sequencing the PCR products and showing that the 5' LTR flanking sequences of proviral DNA obtained from the different subjects presented no homology, thus suggesting that no specific site or sequence is required for the integration process of HTLV-II. The results indicate that the HTLV-II high proviral load observed in PBMCs from infected patients is associated with a clonal expansion of HTLV-II-infected cells. This study also suggests that the very high genetic stability of HTLV-II could be explained by viral amplification via clonal expansion rather than by reverse transcription.

Complete nucleotide sequence of the Italian human T-cell lymphotropic virus type II isolate Gu and phylogenetic identification of a possible origin of South European epidemics.

The complete nucleotide sequence of a human T-cell lymphotropic virus type II isolate (HTLV-II-Gu) from an Italian injecting drug user was obtained, representing the first entire sequence of a European HTLV-II isolate. The HTLV-II-Gu genome was more similar to the HTLV-IIb-NRA isolate (98.4%) and HTLV-IIb-G12 (98.2%) than to HTLV-IIa-Mo (95.2%). The classification of HTLV-II-Gu as subtype IIb was confirmed by restriction analysis. Just as for HTLV-IIa strain Mo, HTLV-IIb-Gu cultured lymphocytes produce two additional mRNAs generated through alternative splicing in the pX region. A phylogenetic analysis was performed by using the methods of neighbour-joining and parsimony with bootstrapping, and maximum likelihood. The different gene regions were analysed separately, comparing Gu with all other HTLV-II strains presently available. In the LTR, as well as in other genome regions, a clear separation between IIa and IIb was evident, and within the IIb subtype three clusters were present of which two were well supported; one contained exclusively Amerindian strains and the other included all Italian and Spanish strains together with two strains obtained from New York drug users. All data clearly showed that HTLV-IIa and IIb subtypes are closely related and are equidistant from HTLV-I, suggesting that both groups evolved simultaneously. The results suggest that HTLV-II-Gu and other IIb South European isolates were probably derived from North American IIb isolates. The data also indicate that sequence analysis is necessary to further classify IIa and IIb subtypes.

Quantification of HTLV-II proviral copies by competitive polymerase chain reaction in peripheral blood mononuclear cells of Italian injecting drug users, central Africans, and Amerindians.

To better correlate the burden of human T cell leukemia virus type I (HTLV-I) and type II (HTLV-II) infection with diagnostic and prognostic markers, we developed a new competitive polymerase chain reaction (PCR) assay for the quantitative determination of proviral copy numbers in infected cells. A competitive plasmid was constructed that carried a 112-bp fragment from a highly conserved region of the HTLV tax gene and that was further modified by inserting a sequence of 24 bp. This competitive PCR assay system can be used for the quantification of HTLV-I and HTLV-II proviral DNA as demonstrated by using HTLV-I- and HTLV-II-infected cell lines and/or patient material. We determined the HTLV-II proviral load in peripheral blood mononuclear cells (PBMCs) of 11 Italian injecting drug users (IDUs) infected by this virus and in PBMCs of 10 seropositive Amerindian and Central African individuals from endemically infected ethnic groups. A great variation was observed in the number of HTLV-II proviral sequences in the PBMCs of Italian drug abusers, ranging from 5-10 to 16,239 copies/10(5) cells. There was no clear-cut correlation between proviral load, CD8 count, stage of HIV-1 infection, and therapy. A considerable variation in HTLV-II proviral load was also observed in PBMCs of Amerindians and Central Africans with no correlation between the amount of HTLV-II provirus and the geographic origin of the infected individuals.

Identification of IIa and IIb molecular subtypes of human T-cell lymphotropic virus type II among Italian injecting drug users.

The molecular characterization of two human T-cell lymphotropic virus type II (HTLV-II) isolates, Gu and Va, obtained from Italian injecting drug users (IDUs) has indicated that these isolates belong to the HTLV-IIb subtype. To establish whether Italian IDUs are also infected by the HTLV-IIa variant, sequencing of the gp21 env gene of proviral DNA from further patients was carried out. Two new isolates, Bo and Md, were found, which presented a divergence of 0.4-0.7% from the IIa prototype HTLV-II-Mo, thus indicating that they belong to the HTLV-IIa subtype. The results strongly support the existence of two distinct molecular subtypes of HTLV-II infecting Italian IDUs and demonstrate that, although the IIb subtype appears to be prevalent, there is the same variability for this virus in Europe as found in the United States. The Italian IIb isolates were also seen to encode an additional 25 amino acids at the C-terminal end of tax protein, as already shown for other IIb isolates. The identification of two HTLV-II molecular subtypes among Italian drug addicts will be useful in tracing the worldwide distribution of this virus and in further understanding its molecular structure and biology.

Prognostic value of adenosine deaminase compared to other markers for progression to acquired immunodeficiency syndrome among intravenous drug users.

This study was designed to determine the prognostic value of erythrocyte adenosine deaminase (ADA) as a possible indicator of progression to AIDS, and compare this with other known cellular and serological markers. At the end of a 3-year study, a cohort of 114 human immunodeficiency virus-1 (HIV-1) seropositive intravenous drug users (IVDUs) from the five different Center for Disease Control (CDC) groups was examined in order to estimate the prognostic relevance with respect to the progression to acquired immunodeficiency syndrome (AIDS) of each of the following markers at baseline value: number and percentage of CD4+ T cells, number of CD8+ T cells, CD4+/CD8+ ratio, IgA and beta 2 microglobulin and ADA levels, and the presence of HIV antigens. Moreover, 57 IVDUs belonging to II and III CDC groups were analyzed in a follow-up study at 6-month intervals, in order to evaluate and compare the behavior of each marker over time. The prognostic significance of each marker was assessed by computing the survival distribution and the Cox analysis in a multivariate model providing the set of markers with greatest predictive value. The levels of ADA and the CD4+/CD8+ ratio showed a linear association with disease staging, whereas beta 2 microglobulin and CD4+/CD8+ ratio were the best predictors for AIDS progression. A highly significant increase in ADA and beta 2 microglobulin was observed during follow-up. The results obtained among HIV-positive IVDUs clearly indicate that the erythrocyte ADA may be considered a reliable marker of the development of HIV infection from the intermediate stages of the disease onwards.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular tropism of human T-cell leukemia virus type II is enlarged to B lymphocytes in patients with high proviral load.

To establish the in vivo cellular tropism of human T-cell leukemia virus type II (HTLV-II) in peripheral blood, subpopulations of mononuclear cells isolated from patients with a history of drug abuse and with high proviral load were analyzed by polymerase chain reaction for the presence of the proviral sequences. After purification of cellular subsets by immunomagnetic fractionation of blood cells of an infected patient, HTLV-II DNA was detected in CD4+ and CD8+ T-cells as well as in CD19+ B-cells. A positive PCR signal was obtained for purified B-cells also at limiting dilutions. This observation was confirmed by purifying the B-cell fraction by a two-step immunomagnetic procedure from the peripheral blood of another patient with very high HTLV-II copy number and quantifying the B-cell proviral load by means of competitive PCR. A proviral copy number of 90/100 B-cells was found, demonstrating that the great majority of these cells were infected by HTLV-II in this subject. The results indicate that HTLV-II has a broad host range in some infected individuals, showing an enlargement of cellular tropism to B lymphocytes and suggesting that this expression is associated with an increase in proviral load.

Molecular characterization of two isolates of human T cell leukaemia virus type II from Italian drug abusers and comparison of genome structure with other isolates.

The human T cell leukaemia virus type II (HTLV-II), whose pathogenicity is as yet unclear, was recently found to be associated with intravenous drug abuse in North America and Europe. HTLV-II was isolated from two Italian drug abusers belonging to the same cohort and coinfected with human immunodeficiency virus type 1. Two new isolates, HTLV-II Gu and Va, were established in a culture of BJAB cells, a continuous B cell line (Epstein-Barr virus-negative), and characterized by nucleotide sequence analysis of the long terminal repeat (LTR) and portions of the gag, env and X regions. These sequences were compared to those of the HTLV-II Mo isolate reported in the literature. No major variations were observed in important regulatory elements of LTR nor in the stem-bulge-loop configuration known to be essential for binding of rex protein. The results obtained from the sequence of the 1988 nucleotides examined indicated a 1.6% variability between the Gu and Va isolates and about 6% with respect to Mo. Notable differences were found in the structure of putative open reading frames of the X region when compared to those reported for the Mo isolate. Restriction analysis of proviral DNA of two isolates and comparison with the physical map of the Mo isolate confirmed the existence of genetic heterogeneity in the HTLV-II group and demonstrated that the new isolates Gu and Va belong to the HTLV-IIb subtype. The results of this study show that the new isolates have distinct features with respect to the Mo isolate though all important regulatory elements of the LTR appear to be well conserved.

Contractile effect of endothelin-2 on the isolated human saphenous vein.

The contractile effect of endothelin-2 was investigated in isolated human saphenous vein preparations. Spare segments taken from revascularized patients were set up in isolated organ chambers and mechanical activity was recorded under isometric conditions. Endothelin-2 (10(-11)-10(-7) M) evoked a dose-dependent contractile response, having the same efficacy as noradrenaline and 100 times its potency. Conversely, the "selective" ETB agonist, C-terminal hexapeptide endothelin (16-21), was completely ineffective. The activity of endothelin-2 was not modified by phentolamine, saralasin and indomethacin, thus excluding a direct or indirect activation of alpha-adrenoceptors and angiotensin receptors as well as the synthesis of cyclooxygenase products. Calcium removal from nutrient fluid depressed, but not fully abolished, the contractile effect of endothelin-2; furthermore, calcium channel blockers, verapamil and nifedipine, produced only a partial inhibition of the endothelin-2-induced contractions. These observations suggest that endothelin-2 induces a direct activation of specific receptors in the saphenous vein muscle and that both intracellular and extracellular calcium pools may be involved in the contractile effect of the peptide.