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Chloracne, also known as metabolizing acquired dioxin-induced skin hamartomas (MADISH), is a rare disfiguring disease related to dioxin exposure. There is a paucity of literature on the clinical manifestations and pathogenesis of chloracne/MADISH. The aim of this study was to assess the clinical features of this very unusual acneiform eruption and to explore the pathogenesis of the disease. This was a retrospective, observational report study was conducted on five patients belonging to the same nuclear family (father, mother and three children) and a relative (father's brother) living in the same house. Histopathological, immunohistochemical, laboratory and toxicological analyses were performed for all patients. The results suggest that CYP1A1 in human skin is a diagnostic biomarker in chloracne, and was positive for all the patients in our sample. Tetrachlorodibenzo-p-dioxin is the most investigated dioxin responsible for chloracne; however, several other agonists, whether dioxin-like or not, can activate the aryl hydrocarbon receptor. To our knowledge, this Italian case series is the first study to suggest polychlorinated biphenyls as a possible cause of an overstimulation of aryl hydrocarbons causing the consequent acneiform eruption.

This Italian case series has shown that PCBscould promote an overstimulation of AhR in the skin, favouringthe consequent acneic eruption. A combined dermatologic andmetabolic therapy can lead to a complete healing of chloracne

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BACKGROUND: Anti-nuclear antibodies (ANA), anti-extractable nuclear antigens (ENA) and anti-dsDNA antibodies are often associated with cutaneous lupus erythematosus (CLE), with variable frequency depending on skin subtype. However, specific data based on large case-series on the pathogenetic, diagnostic and prognostic meaning of such autoantibodies are still lacking. OBJECTIVE: To characterize the correlations between CLE subtypes as well as LE-non-specific skin lesions and their autoantibody pattern. METHODS: Epidemiological, clinical and immunopathological data of 619 Italian patients with CLE and LE-non-specific skin lesions were analysed. Differences in age, sex, clinical features and autoantibody profile were evaluated in each LE subgroup. RESULTS: Anti-nuclear antibodies (P < 0.0001), anti-dsDNA (P < 0.0001), ENA (P = 0.001), anti-Sm (P = 0.001), anti-RNP (P = 0.004) and anti-histone (P = 0.005) antibodies were associated with SLE. A strong association between ANA (P < 0.0001) and anti-dsDNA (P < 0.0001) and female gender was also found: positive ANA and positive anti-dsDNA had a higher prevalence among females. Chronic CLE resulted to be negatively associated with ENA (OR = 0.51, P < 0.0001), anti-Ro/SSA (OR = 0.49, P < 0.0001) and anti-dsDNA (OR = 0.37, P < 0.0001). Intermittent CLE resulted to be negatively associated with ENA (OR = 0.50, P = 0.007) and ANA (OR = 0.61, P = 0.025). Subacute CLE resulted to be associated with ENA (OR = 5.19, P < 0.0001), anti-Ro/SSA (OR = 3.83, P < 0.0001), anti-Smith (OR = 2.95, P = 0.004) and anti-RNP (OR = 3.18, P = 0.007). Acute CLE resulted to be strongly associated with anti-dsDNA (OR = 6.0, P < 0.0001) and ANA (OR = 18.1, P < 0.0001). LE-non-specific skin lesions resulted to be significantly associated with systemic involvement. Livedo reticularis was significantly associated with ENA (P = 0.007) and anti-Ro/SSA (P = 0.036). Palpable purpura and periungual telangiectasia were significantly associated with ANA. CONCLUSION: According to our findings, some well-known associations between CLE subtypes and autoantibody profile were confirmed; moreover, specific association between autoantibodies and LE-non-specific skin lesions was highlighted. A strict association between anti-ENA and anti-Ro/SSA antibodies and livedo reticularis, ANA and palpable purpura, and ANA and periungual telangiectasia was evidenced.

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Background and aims We assessed post-marketing safety of sodium-glucose co-transporter-2 inhibitors (SGLT2-Is) by analyzing adverse events (AEs) reported in international pharmacovigilance databases. Methods and results Eudravigilance, WHO-Vigibase (as of Feb 25, 2017) and the FDA Adverse Event Reporting System (FAERS, from 2004 to 2016 second quarter) were queried to extract AEs recording SGLT2-Is as suspect. Disproportionality analyses (case/non-case method) were performed in FAERS by calculating the reporting odds ratios (RORs) from System Organ Classes (SOCs) to Preferred Terms (PTs) (precise clinical entities). Potential signals were defined by statistically-significant ROR (lower limit of the 95% confidence interval – LL95%CI – >1) undetected by literature analysis (as of December 2016). SGLT2-Is were recorded in 7972, 19,775, 11,137 reports (Eudravigilance, WHO-Vigibase and FAERS, respectively); in FAERS, statistically significant ROR emerged for the following SOCs: “infections and infestations” (N = 2162; LL95%CI = 3.25), “metabolism and nutrition disorders” (2278; 1.36), “renal and urinary disorders” (1665; 2.31), “reproductive system and breast disorders” (471; 4.85), “skin and subcutaneous tissue disorders” (1136; 1.52). Skin toxicity emerged as potential signal (e.g., rash, photosensitivity, urticaria as PTs), both for SGLT2-Is as a class and as individual drugs. Severe adverse skin events (81 reports, 7% of the skin cases) mainly occurred in females aged 18–65 using SGLT2-Is as single antidiabetic regimen. Conclusion Among antidiabetics, SGLT2-Is are associated with higher reporting of infections, metabolism, renal and reproductive AEs, corroborating clinical trial evidence. Their large reporting patterns and the unexpected signal of skin toxicity justify active vigilance by clinicians and “real-time” monitoring by pharmacovigilance experts.

Background: Parvovirus B19 is the aetiological agent of erythema infectiosum. The presence of B19 DNA in lesional skin of other cutaneous manifestations has frequently been reported although there is disagreement on the role of the B19 virus in tissues. Objectives: To investigate the presence of B19 DNA (1) in skin lesions of patients with a described B19-related disease, (2) in skin lesions of B19-unrelated diseases and (3) in healthy skin. Methods: A total of 121 skin samples were examined for the presence of B19 DNA by PCR assays and peptide-nucleic-acid-based in situ hybridisation techniques. Results: B19 DNA was detected in 11/38 (28.9%) pityriasis lichenoides, 8/30 (26.7%) melanocytic naevi, 5/29 (17.2%) primary melanomas and 6/24 (25.0%) healthy skin biopsies. A difference in B19 DNA prevalence was observed in specimens grouped according to age, irrespective of pathologies. Conclusions: B19 DNA can be found in skin tissues of patients with pityriasis lichenoides as well as in lesions not related to B19 infection and in healthy controls. B19 DNA can be detected in skin of young subjects in a significantly high rate compared to adults, suggesting that viral persistence may be the usual outcome after primary infection.

Letter to the Editor

The study evaluated the development of drug resistance in a group of HIV-1 patients. After failure to respond to previous therapy with two non-nucleoside reverse transcriptase inhibitors (NNRTIs), as assessed by the presence of a rebound in viral load or a constant high level of HIV plasma viraemia, the patients were treated with a combination of stavudine, lamivudine and efavirenz (EFV). Results showed that viruses carrying primary mutations, usually K103N, T215Y and M41L, presented higher levels of HIV-1 RNA, suggesting an association between a precise mutation pattern and treatment failure.

Not available.

Background: The efficacy of a specific humoral response to transactivating Tat protein was studied in a group of HIV-1 seropositive drug addicts, who had previously received a similar course of anti-retroviral treatment with two reverse transcriptase inhibitors. Objectives: The aim of the study was to evaluate the meaning of an immune response to Tat protein in HIV-1 seropositive patients with different levels of HIV-1 RNA viremia. Study design: The study analyzed the presence of anti-Tat antibody reacting either with full-length Tat or with individual overlapping Tat-peptides (Tat6–14, Tat11–24, Tat36–50, Tat46–60, Tat56–70 and Tat65–80), in a group of HIV-1 seropositive subjects with different peripheral blood viral loads. Plasma samples were examined by immunoenzymatic assay for the presence of anti-Tat IgG antibody and for the quantification of peripheral blood (plasma) viral load by branched DNA assay. Results: The large majority of HIV-1 patients showed detectable levels of serum IgG to full-length-Tat, and the anti-Tat antibody level presented an inverse correlation with viral load magnitude. The analysis of antibody levels against individual overlapping Tat-peptides clearly showed that an undetectable viral load was significantly associated with the presence of a high antibody concentration against Tat6–14, Tat36–50 and Tat46–60 (P=0.002, P=0.027 and P

Summary Background The vast majority of studies aimed at detecting human papillomavirus (HPV) DNA in skin cancer have used sensitive polymerase chain reaction (PCR) methods but the PCR technique, despite its high sensitivity, is not suitable to ascertain whether (i) the presence of HPV can be related only to few cells harbouring the virus, (ii) the presence of HPV is due to a tumour surface contamination and (iii) the presence of HPV is localized in cancer cells, rather than in normal keratinocytes present in the tumour biopsy. In a recent work we have found mucosal high-risk (HR) HPV genotypes in primary melanoma by PCR. Objectives To localize mucosal HR-HPV nucleic acids and tumoural melanocytic marker in the same sections of primary melanoma samples in order to understand the relationship between HPVs and melanoma cells. Methods We have developed a very sensitive method that combines an enzyme-amplified fluorescent in situ hybridization (ISH) for the detection of HPV nucleic acids (types 16 and 18) with a chemiluminescent immunohistochemistry (IHC) method for the detection of the tumoural melanocytic marker HMB-45 sequentially in the same section. Digital images of fluorescent ISH and chemiluminescent IHC were separately recorded, assigned different colours and merged using specific software for image analysis. Results The combined fluorescent ISH and chemiluminescent IHC demonstrated a sharp colocalization (in the range 60–80%) of HPV nucleic acids and melanoma marker inside the same sections of melanoma biopsies, with a strong specificity and sensitivity. Conclusions The strong colocalization of mucosal HR-HPV nucleic acids and HMB-45 melanocytic marker emphasized that viral nucleic acids were specifically present in melanoma cells and supported a possible active role of HPV in malignant melanoma.

In human immunodeficiency virus type-1 (HIV-1) infected individuals, CD34+ hematopoietic stem/progenitor cells are profoundly impaired in their proliferation/differentiation capacities. The bulk of the available experimental evidence seems to indicate that hematopoietic progenitors are not susceptible to HIV-1 infection and their defects seem rather the consequence of direct or indirect negative influences of HIV-1-specific soluble proteins released by productively infected accessory cells. We have now shown that in the presence of a concurrent human herpesvirus-6 infection, two hematopoietic (TF-1 [erythromyeloid] and KG-1 [lymphomyeloid]) progenitor cell lines and human CD34+ hematopoietic progenitors isolated from the bone marrow of normal donors, became susceptible to HIV-1 infection and permissive to HIV-1 replication, although with a limited virus yield. These results suggest a further possible mechanism leading to hematopoietic derangement in HIV-1-infected subjects and may help to clarify the controversial issue of the susceptibility of human hematopoietic progenitors to HIV-1 infection.

AIMS--To evaluate the presence of cytomegalovirus (CMV) DNA in the cerebrospinal fluid of patients with AIDS and suspected viral encephalitis using an in situ hybridisation assay with digoxigenin labelled CMV DNA probes. METHODS--The presence of CMV DNA was evaluated in cerebrospinal fluid cells of 10 patients with AIDS using in situ hybridisation. The positivity of CMV DNA was confirmed by the presence of CMV induced antigens in the same specimens. The presence of CMV DNA and CMV induced antigens was also analysed in peripheral blood leucocytes. The time required to perform the in situ hybridisation assay was about eight hours. RESULTS--The in situ hybridisation assay was sensitive, specific, and provided good resolution. Six patients proved positive for the presence of CMV DNA in CSF cells and all six also proved positive for CMV DNA in blood leucocytes. Of the six CMV positive patients, five were treated with specific antiviral drugs: of these, one died during the treatment while four clinically recovered after one month of treatment. CONCLUSIONS--The in situ hybridisation assay using digoxigenin labelled CMV DNA probes can be used as a valuable diagnostic test for the detection of CMV DNA in the cerebrospinal fluid cells of patients with suspected CMV encephalitis and can therefore prompt adequate antiviral therapeutic intervention.

We investigated the expression of CD4 antigen in normal bone marrow (BM) samples, enriched in CD34+ hematopoietic progenitor cells. At flow cytometry, a significant fraction (ranging from 25% to 65%) of CD34+ cells also showed low levels of CD4 antigen on their surface. The CD4 receptor densities on the surface of hematopoietic progenitors was approximately 50% that of peripheral blood monocytes and 5% of peripheral blood T lymphocytes. In immunoprecipitation experiments, the CD4 antigen expressed by BM hematopoietic progenitors appeared to be the same form expressed by mature peripheral blood CD4+ cells and appeared to be a potentially functional receptor for human immunodeficiency virus-type 1, because it specifically bound recombinant envelope gp120. Moreover, BM samples, highly enriched in CD34+ cells, showed the presence of CD4 mRNA at reverse transcription- polymerase chain reaction examination. In experiments of complement- mediated cytotoxicity with Leu3-a+Leu3b anti-CD4 monoclonal antibody, a significant reduction in the number of both classes of megakaryocyte (burst-forming unit-meg [BFU-meg] and colony-forming unit-meg [CFU- meg]) and granulocyte/macrophage (CFU-GM) progenitors was observed, whereas erythroid (BFU-E) progenitors were only slightly affected. Moreover, purified CD4+ BM cells obtained by immunomagnetic selection, using high concentrations of Leu3a+Leu3b, showed a colony-forming ability of megakaryocyte and granulocyte/macrophage progenitors comparable with that of CD4- BM cells. In conclusion, the present data show that immature hematopoietic progenitor cells express low levels of CD4, the high-affinity receptor of human immunodeficiency virus-type 1.

Besides a progressive depletion of CD4+ T-lymphocytes, other peripheral blood cytopenias, (granulocytopenia, anemia and thrombocytopenia) are frequently observed in HIV-1 seropositive individuals, especially in patients with overt AIDS. Various experimental evidences suggest that HIV-1 could play a direct role in the pathogenesis of HIV-1 related peripheral blood cytopenias, affecting the survival/proliferation capacity of hematopoietic progenitors. CD34+ human hematopoietic progenitors, however, are substantially not susceptible to HIV-1 infection either in vitro and in vivo and their defects seem rather related to an alteration of bone marrow and peripheral blood microenvironments due to the presence of soluble HIV-1 specific products.

The effect of increasing concentrations (from 0.01 to 10 micrograms/ml) of HIV-1 envelope glycoproteins gp160, gp120, gp41 and core protein p24 was evaluated on the in vitro growth of enriched hematopoietic progenitors (CD34+ cells). Both gp120 and gp160, at concentrations from 0.01 to 10 micrograms/ml, caused a progressive and significant (p less than 0.05) decrease in viable CD34+ cell count in liquid cultures supplemented with 2 ng/ml of human recombinant (r) interleukin-3 (IL-3), evaluated by means of Trypan-blue exclusion and [3H]thymidine ([3H]TdR) incorporation. In the absence of rIL-3, no inhibitory effects were observed even at the highest gp160 and gp120 concentrations explored (10 micrograms/ml). On the contrary, gp41 and p24 did not affect the number of viable CD34+ cells, either in the presence or in the absence of rIL-3. Moreover, gp160 and gp120, but not gp41 and p24, significantly (p less than 0.05) inhibited the in vitro growth of granulomacrophage progenitors (CFU-GM) in a dose-dependent fashion. These data clearly demonstrate that HIV-1 envelope glycoproteins inhibit the growth of purified hematopoietic progenitors. We propose that HIV-1 can impair hematopoiesis through the interaction of gp120/gp160 with CD34+ cell surface, independently of an infectious process.

Seventy infants born to human immunodeficiency virus type 1 (HIV-1) seropositive mothers were studied for specific antibody (IgA, IgM and IgG) production and the presence of active infection (detectable level of virus in peripheral blood lymphocytes). Among these children, followed for up to 15-40 months after birth, 11 presented unequivocal signs of HIV-1 infection (persistent p24 antigenemia and/or positive virus isolation). Analysis of sera by immunoblotting showed that IgA antibody to HIV-1 p24 core protein, alone or associated with envelope glycoproteins (gp120, gp41), was present in the majority of infected babies (7 of 11), while IgM was found in a lower percentage of cases (4 of 11). No IgA and or IgM antibody to HIV-1 was ever found in babies, born to seropositive mothers, who seroreverted after birth or in the control group enrolled in this study. Our results indicate that immunoblotting analysis of IgA antibody to HIV-1 polypeptides may represent a useful complementary prognostic marker in children born to HIV-1 seropositive mothers.

We compared cytomegalovirus (CMV) isolation in tissue culture and viral DNA detection by DNA:DNA hybridization in 60 clinical urine samples concentrated by different procedures. Our results showed that urine concentration by filtration is the easiest and quickest procedure allowing further detection of CMV. With optimized working conditions, the CMV detection in urine can be done in 5 hr without requiring cell cultures or costly instruments.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are considered to be severity variants of the same disease, which is almost always associated with drug intake. In contrast, erythema multiforme (EM) is a disorder regarded as only rarely caused by drugs. Keratinocyte apoptosis has been shown to play an important part in the pathogenesis of SJS and TEN, whilst its role in EM remains controversial. To determine the expression of apoptosis-associated molecules Fas, Fas ligand (FasL), Bcl-2 and Bax in the above disorders, an immunohistochemical analysis was performed. We studied both lesional skin from thirty patients having drug-induced EM and 5 cases classified within the SJS/TEN spectrum and normal skin samples. We found a keratinocyte overexpression of Fas antigen, an important molecule mediating apoptosis, not only in SJS and TEN but also in EM. Another noteworthy finding was the strong expression of Bcl-2, a protein known as blocking apoptosis, along the basal layer and in the dermal infiltrate both in SJS/TEN and in EM. Taken together, these findings suggest that Fas-dependent keratinocyte apoptosis may play a part in the pathogenesis of both SJS/TEN and EM. Fas-mediated cell death may be partially suppressed by the Bcl-2 protein. Copyright © by Biolife, s.a.s.

The aim of this pilot phase II trial was to investigate the toxicity and anti-tumour activity of a novel metronomic regimen of weekly cisplatin (CDDP) and oral etoposide (VP16) in high-risk patients with advanced NSCLC. The study enrolled 31 high-risk patients (27 men and 4 women aged 16-82 years; mean, 64.3) with NSCLC (18 stage IIIB and 13 stage IV) and an ECOG performance status of < or = 3, all of whom received weekly CDDP 30 mg/m2 iv on days 1, 8, 14 and 28 of each cycle and oral daily etoposide 50 mg/m2 on 21 of the 28 days. The most frequent adverse events were grade III leukopenia and anemia; nevertheless, three patients died of pulmonary embolism after 2, 3 and 6 weeks of treatment. The objective response (OR) rate was 45.2% (2 complete and 12 partial), and the disease control rate was 58.1% (14 ORs and 4 disease stabilisations). The mean time to progression and survival were respectively nine months (95% CI, 6.3-15.8 months) and thirteen months (95% CI, 9.1-20.5 months). Pharmacological analysis showed that this metronomic regimen allows a much greater median monthly area under the curve of CDDP and VP16 than conventional treatment schedules. Our findings also suggest that this treatment schedule may affect tumour growth and neoangiogenesis by changing peripheral blood vascular-endothelial growth factor levels. These preliminary results indicate that our metronomic regimen is well tolerated and active, even in patients with a very poor prognosis.

A 1.8 V 256 Mb 2b/cell NOR flash memory is designed in a 130 nm technology. A fast gate-voltage-ramp constant-current-reading concept is implemented to obtain a robust read-while-write/erase function and 125 MHz burst read frequency.

Summary Background Some studies have shown that cutaneous and mucosal melanoma biopsy specimens harbour human papillomavirus (HPV), suggesting that this virus may play a role in development and progression of the tumour. Objectives To investigate the presence of HPV DNA and the prevalence of different high-risk mucosal HPV genotypes in primary melanoma (PM) and in acquired dysplastic melanocytic naevi (ADMN). Methods Fifty-one PMs from 18 men and 33 women (median age 55·5 years), 33 ADMN from 15 men and 18 women (median age 35·1 years) and 20 control skin samples from nine men and 11 women (median age 43·5 years) were studied. All diagnoses were made after histological analysis. HPV DNA analysis was made using two different polymerase chain reaction–enzyme-linked immunosorbent assay (PCR–ELISA) methods, namely MY-PCR and GP-PCR. Results Using GP-PCR, mucosal HPVs were detected in 14 PMs (27%; P = 0·0166) and eight ADMN (24%; P = 0·0367), while with MY-PCR, mucosal HPVs were found in 11 PMs (22%; P = 0·04) and five ADMN (15%; P not significant). All control skin samples were negative for mucosal HPVs with both DNA amplification procedures. Conclusions Using our PCR–ELISA methods, the detection of mucosal high-risk HPV genotypes in 24% of precursor lesions (ADMN) and in 27% of PMs adds to the body of evidence indicating a colocalization of mucosal HPV and tumoral melanocytic pathologies.

A genotyping assay was used to define human immunodeficiency virus type 1 (HIV-1) reverse transcriptase codons in plasma samples from 80 HIV-1 patients extensively treated with two nucleoside reverse transcriptase (zidovudine and lamivudine) and one non nucleoside reverse transcriptase (nevirapine) inhibitor. The frequencies of T215S/Y/F, M41L, D67N, L210W K70R, K219Q mutations, detectable in plasma samples, conferring resistance to zidovudine were 61.2, 56.2, 36.2, 31.5, 27.5 and 17.5%, respectively. Mutations (M184V or M184I) conferring resistance to lamivudine were detected in an extremely high percentage of patients (61%). Among mutations correlated to high (K103N, V106A, Y181C/I, Y188C/H/L, G190A/C/E/Q/S/T) or moderate (V108I, V118I) levels of nevirapine resistance, the predominant amino acid change was a substitution at 103 codon, present in 24 of 80 samples tested. Finally Q151M, the marker mutation able to confer resistance to all nucleoside analogues, was detected in seven patients with a viral load of between 1×10 4 and 9×10 4 HIV-1 RNA copies/ml. The relationship between the genotype and the viral load showed that the incidence of some specific mutations [M41L, T215Y (correlated to zidovudine resistance) and K103N (correlated to all NNRTIs drugs)] significantly ( P =0.001) increased with higher viral load. Our results, albeit limited to a small cohort, showed a high frequency of mutations correlated to drugs in use, suggesting a need for therapeutic change in the near future and demonstrating that the development of genotyping tests helps to guide the therapeutic management of HIV-1 infected people. Our data highlight the dangers of selecting antiretroviral therapy without previous antiretroviral drug testing. Although the cost of these assays is a concern, prescribing inefficacious drugs could create serious problems for HIV-1 patients.

Summary CXCR4 is the high affinity receptor for the SDF-1α chemokine and represents the main coreceptor for HIV-1 T-tropic strains. The surface expression of CXCR4 was analysed in CD34+ haematopoietic progenitors, induced to differentiate along the erythroid or granulocytic lineages, in liquid cultures supplemented or not with HIV-1 Tat protein. At concentrations as low as 1–10 ng/ml, synthetic Tat protein significantly increased the surface expression of CXCR4 in erythroid but not in granulocytic cells. The Tat-mediated up-regulation of surface CXCR4 was accompanied by a concomitant increase of CXCR4 mRNA and total CXCR4 protein content in cells developing along the erythroid lineage after 6–10 days of culture. Moreover, addition of SDF-1α (200 ng/ml) induced a significant higher rate of apoptosis in Tat-treated erythroid cells in comparison with control cells. These results demonstrated for the first time a direct positive role in haematopoietic gene regulation of Tat protein, and suggest the possible involvement of Tat in HIV-1-induced anaemia.

We retrospectively studied 132 patients with dermatomyositis; 84 had idiopathic, 30 paraneoplastic, 5 juvenile and 13 amyopathic forms of the disease. The commonest features were macular erythema, heliotropic erythema and Gottron's papules. Flagellate erythema occurred in 5% of patients with idiopathic dermatomyositis and correlated with the disease activity. Necrotic lesions were also found in this group of patients but did not always signal malignancy. The prevalence of malignancy was high (23%). Raynaud's phenomenon occurred in 10.6% of patients, also in those with malignancy. Dysphagia, interstitial lung disease and arthralgias affected 20%, 8% and 40% of patients, respectively. Anti-Jo-1 antibodies were found in 5% of patients with idiopathic dermatomyositis and low titre ANA in 1/3 of patients. ANA did not correlate with the disease activity. We confirmed the data from the literature, but no cutaneous sign, constitutional symptom or circulating antibody was found marking a particular subtype of the disease.

As the emergence of highly resistant virus might compromise antiretroviral regimens in HIV-1 infected patients, a constant analysis of genotypic mutations should be performed to establish the magnitude of mutation prevalence and gauge their impact in patients treated extensively with combination therapy. The frequency of multiple dideoxynucleoside analogue resistance (MddNR) was evaluated in a group of Italian HIV-1 seropositive patients who failed to respond to therapy despite a long-lasting drug treatment. Results showed the presence of one or more mutations (A62V, V75I, F77L, F116Y and Q151M) able to confer resistance to all NRTIs in a relatively high percentage (7.9%) of patients enrolled in the study. Moreover, a significantly lower HIV-1 viral replication in patients with MddNR, suggested the importance of monitoring HIV-1 subjects not only by viral load, but also by drug resistance testing, so that a correct drug regimen may be chosen.

The broad spectrum of clinical manifestations associated with B19 parvovirus often allows the infection to go unrecognized. We tested for the presence of B19 parvovirus and the specific serological response in serum from hospitalized patients submitted for viral investigations without any specific indications for B19 parvovirus. We diagnosed human parvovirus B19 infection in four paediatric patients showing different clinical manifestations. The patients, aged between 5 and 8 years, were admitted to hospital for: (1) petechial rash; (2) mononucleosis-like syndrome; (3) neurological syndrome; and (4) respiratory disease (in an immunodeficient patient).

The efficacy of highly active antiretroviral therapy (HAART) was prospectively monitored in HIV-1 seropositive patients by analyzing HIV-1 RNA viral load.The aim of the study was to evaluate the viral load course in two different groups of patients (group 1: CD4400/mm(3), group 2: CD4200/mm(3)) during a period ranging from 9 to 25 months.HIV-1 viral load, at the start and during HAART, was analyzed in 117 patients who had previously been treated only with two anti-transcriptase drugs but were naive for protease inhibitors.The results showed that, after the beginning of therapy, high plasma HIV-1 RNA levels dropped to undetectable values (50 copies HIV-RNA/ml) in one third of patients over a mean period of about 9 months irrespective of the initial CD4 cell count, even though a viral reduction of at least 2log(s) in a significantly shorter period of time (P0.001) was observed only among patients who began retroviral therapy with a higher CD4 cell count.The response to HAART was not dramatically affected by the initial CD4 count. Though restricted to a small number of subjects, the data support the idea that therapeutic intervention can be effective even in an advanced stage of HIV-1 infection, when patients show a decreased number of CD4 T-lymphocytes.

We have previously demonstrated that the addition in culture of recombinant HIV-1 IIIB envelope gp120 affects the survival/growth of pluripotent haemopoietic progenitors, and, in particular, of those committed towards the megakaryocytic lineage. To characterize some of the molecular mechanisms involved in this phenomenon, we investigated the expression of members of the activating protein-1 (AP-1) complex in the HEL megakaryoblastic cell line. Following the treatment of HEL cells with recombinant IIIB envelope gp120, we noticed: (i) increased levels of endogenous c-fos and c-jun mRNA and proteins, (ii) activation of both c-fos and c-jun promoters, and (iii) a very rapid stimulation of a MAPK/ERK pathway.

Infections of the oral cavity are most commonly odontogenic in origin and include dental caries, periapical infections, gingivitis, periodontal infec-tions and abscesses, and deep fascial space infec-tions. Although rare, life-threatening complications, such as mediastinal or intracranial extension, retro-pharyngeal spread and airway obstruction, pleuropulmonary suppuration, and hematogenous disse-mination to heart valves, prosthetic devices, and other metastatic foci are known, which indicate the potentially serious nature of these infections.

A dot blot hybridization immunoenzymatic assay for the rapid detection of cytomegalovirus DNA in urine samples was developed by using a digoxigenin-labeled probe which was immunoenzymatically visualized by antidigoxigenin Fab fragments labeled with alkaline phosphatase. A total of 516 urine samples from different groups of subjects were analyzed, and the hybridization assay was able to yield results within 24 h. The results obtained were compared with results for detection of cytomegalovirus antigens in infected cell cultures.

Haematopoietic progenitor cells (HPC) of HIV-1-infected patients are severely compromised in their replication and clonogenic capacities, and show an enhanced propensity to apoptosis, despite the lack of productive or latent HIV-1 infection.To investigate telomerase enzyme levels in CD34+ HPC isolated from HIV-1-infected patients, because the absence of telomerase activity has been found to be correlated with a diminished replication potential.Telomerase levels were measured by a PCR-based telomeric repeat amplification protocol. CD34+ HPC isolated from the peripheral blood of 11 HIV-1-infected patients were compared with CD34+ HPC isolated from peripheral blood (nine subjects) or bone marrow (six subjects) from 15 healthy donors. Telomerase levels were also studied in normal HPC after exposure to either gp120 or transforming growth factor (TGF)-beta1.CD34+ HPC isolated from either peripheral blood or bone marrow from healthy donors expressed a high level of telomerase activity. On the contrary, CD34+ HPC isolated from HIV-1-seropositive patients did not express any detectable telomerase activity in nine patients, and a clearly reduced enzymatic activity in two patients. Furthermore, telomerase activity in normal CD34+ HPC exposed to recombinant gp120 was significantly reduced, and to a higher extent than in CD34+ HPC exposed to recombinant TGF-beta1.This is the first study to demonstrate severely impaired telomerase activity in uninfected CD34+ HPC isolated from HIV-1-infected patients. The mechanism underlying this impairment probably involves the interaction of HIV-1 envelope glycoprotein gp120 with the cell membrane. These results may add to our understanding of the pathogenesis of the lesion of the HPC compartment.

CD3 mAb and HIV-1 Tat protein co-immobilized on plastic were able to induce a strong proliferation of resting human CD4 T cells, cultured in a serum-free chemically defined medium. Blocking studies performed with heparin or peptides containing the RGD sequence demonstrated that the heparin-binding basic domain of Tat plays a predominant role in CD4+ T cell activation. Moreover, the enhanced proliferative response of CD4+ T cells to immobilized Tat appeared to be mediated by alpha 5, beta 1, and alpha v subunits of surface integrin receptors. In contrast, soluble Tat showed a dose-dependent inhibitory activity on the proliferative response of resting CD4+ T cells stimulated by CD3 mAb co-immobilized with Tat or fibronectin, but not with CD28 mAb. In transient transfection assays performed with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) plasmid CD3 mAb co-immobilized with Tat or fibronectin or CD28 mAb significantly stimulated CAT activity over the background. On the other hand, while immobilized Tat alone had no effects on LTR transactivation, soluble Tat was able to transactivate LTR-CAT in a dose-dependent manner. When CD4+ T cells activated by CD3 mAb co-immobilized with Tat were recovered, cultured for 7 days with 25 U/ml recombinant IL-2, and given an additional activation signal by recross-linking CD3 mAb, a marked increase of apoptosis was observed with respect to cells not subjected to CD3 mAb recross-linking. While co-immobilized Tat plus CD3 mAb did not show any significant effect on activation-induced cell death, high concentrations of soluble Tat synergized with immobilized CD3 mAb in the induction of apoptosis.

Background and Objectives: The ligase chain reaction is an in vitro DNA amplification technique that exponentially amplifies selected DNA sequences. Goal: To evaluate a ligase chain reaction assay for the detection of Chlamydia trachomatis cryptic plasmid DNA (LCx Chlamydia) in patients routinely attending a sexually transmitted disease center in Italy. Study Design: Urethral or cervical swabs were obtained from 501 consecutive patients (334 men and 167 women). The samples were assayed in parallel with LCx Chlamydia and conventional tissue culture; discordant results were further assayed by direct immunofluorescence and a ligase chain reaction with alternate primers. Results: After resolution of discordant results, the LCx method showed a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 99.3%, 96.7%, and 100% in men; 100%, 100%, 100%, and 100% in women; and 100%, 99.5%, 97.1%, and 100% overall, respectively. By comparison, the sensitivity of tissue culture was 81.4% in men, 50% in women, and 77.6% overall. Conclusions: The automated LCx method is sensitive, fast, and accurate and represents a useful diagnostic tool for C. trachomatis infection, even in low and medium prevalence populations.

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We report a methodology for detecting specific DNA sequences directly inside cells, combining in situ PCR and flow cytometry. This technique is based on in situ PCR performed in the presence of digoxigenin-labeled dUTP to obtain a digoxigenin-labeled amplicon, which is then revealed by an anti-digoxigenin polyclonal antibody directly conjugated to fluorescein. Fluorescence intensity is next evaluated by flow cytometry. Our experimental models were represented by the lymphoblastoid cell lines 8E5LAV, carrying an integrated HIV-1 DNA proviral copy per cell, and A.301, infected in vitro with HIV-1 (strain IIIB). The technique is described in detail with particular attention to the optimization of critical fixation and permeabilization steps. This method allows not only the detection but also an accurate quantification of the number of positive cells in a background of negative cells. Moreover, it has the potentiality to develop into a multiparametric method for the simultaneous study of specific DNA or RNA sequences and surface or intracellular markers.

Human immunodeficiency virus type 1 (HIV-1) transactivating Tat protein is pivotal to virus replication. Tat's potential effects on HIV-1 pathogenesis, however, go well beyond its role in the virus's life cycle. Current data indicate that biologically active Tat is released from HIV-1-infected cells and readily endocytosed and targeted to the nucleus of nearby, or perhaps distant, cells, where it may exert a series of pleiotropic effects. This paracrine action has been extensively investigated, and depending on the amounts of exogenously added Tat, its effects may extend from the suppression of immunocompetent cells to transactivation of heterologous genes to the promotion of growth of Kaposi's sarcoma spindle cells. We have already observed that various cell lines, either permanently transfected with an expressive HIV-1 tat gene construct or cultured in the presence of exogenously added Tat protein, are protected from programmed cell death after serum withdrawal or other apoptotic stimuli. The present article shows that various types (lymphoblastoid, epithelial, neuronal) of permanently tat-transfected cell lines actively release fully bioactive Tat protein. The addition of anti-Tat antibody to the culture medium completely abolishes their increased survival/proliferation capacity in serum-free culture. In these conditions, therefore, the enhanced survival/proliferation potential of permanently tat-transfected cells seems entirely dependent on a Tat-protein autocrine loop. The finding that anti-Tat antibody, added to culture medium, exerts a negative influence on the expression of a Tat-responsive HIV-1 long terminal repeat chloramphenicol-acetyltransferase construct, transiently transfected into permanently tat-transfected cells, suggests that the Tat autocrine loop may also be required for optimal HIV-1 long terminal repeat transactivation.

Members of the dendritic cell (DC) lineage, including Langerhans cells (LC) of squamous epithelia and peripheral blood (PB) DC, have been suggested to play an important role in the pathogenesis of HIV-1 infection1. PB DC from HIV-1 infected patients contain significant amounts of HIV-1 proviral DNA and can show viral budding1. Human epidermal LC express CD4 molecules on their membrane and are thus a likely target of HIV infection. We have demonstrated that purified preparations of epidermal LC, but not other epidermal cell (EC) types, harbor HIV-1 proviral DNA and RNA, indicating that LC are productively infected and can serve as a reservoir of the virus2,3. Moreover, the HIV DNA copy number in LC of AIDS patients is comparable to that found in PB CD4+ T cells of patients at the same disease stage3. PB DC from healthy seronegative donors have been infected in vitro with HIV and could transmit the infection to other cells of myeloid origin4. Few studies have addressed the in vitro infection of epidermal LC. LC possess membrane gpl20-binding sites5; in addition, Berger and co-workers have been able to infect in vitro both LC histiocytosis cells and LC-enriched EC, but the nature of the EC type infected could not be determined6.

Human CD4+ T lymphoblastoid Jurkat cells were stably transfected with two different plasmid vectors containing the cDNA of human immunodeficiency virus-type 1 (HIV-1) tat gene under the control of either the promoter of simian virus 40 (pRPneo/tat) or the long terminal repeat region of SL3 murine leukaemia virus (pRPneo/SL3/tat). Both pRPneo/tat and pRPneo/SL3/tat Jurkat cell lines showed a constant and high production of bioactive Tat in transient co-transfection assays with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) reporter plasmid. Tat-positive and mock-transfected Jurkat cells were cultured with various cytotoxic agents, which have been associated to the progressive loss of CD4 T-lymphocytes characteristic of HIV-1 disease. In the presence of recombinant tumour necrosis factor-alpha (TNF-alpha), anti-fas antibody, Leu3a anti-CD4 antibody, the percentage of apoptosis, evaluated in a 24-72 h short-term assay, was lower (P < 0.05) in tat-positive Jurkat cells than in mock-transfected controls. The low susceptibility to the cytotoxic activity of TNF-alpha and anti-fas antibody of tat-transfected cells was confirmed by counting viable cells up to 15 d of culture. Also, recombinant Tat protein was able to prevent the increase of apoptosis induced in mock-transfected Jurkat by TNF-alpha. Of note, tat-expressing cells showed a better survival with respect to mock-transfected control cells even when acutely infected with high doses (500,000 cpm of reverse transcriptase) of HIV-1 (strain IIIB) or treated with heat-inactivated HIV-1. These data demonstrate that the expression of the regulatory HIV-1 Tat protein is able to rescue Jurkat lymphoblastoid cells from apoptosis induced by a variety of cytotoxic agents. Since Tat protein expression is restricted to the initial phases of an active HIV-1 replication, the anti-apoptotic effect of Tat could have the physiological significance of selectively protecting HIV-1 producing cells from death, at least for the time necessary to allow virus production and spreading.