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Due to the early diagnosis, melanomas can be diagnosed in early stages. Most melanomas tend not to show morphological criteria of malignancy in the very early stages. They rather resemble benign moles. For patients with hundreds of atypical lesions, follow-up examinations using digital dermoscopy are very helpful. This technique enables the physician to monitor lesions and to detect microscopic change. Lesions with microscopic change are thought to be high risk lesions and should be removed this will represent important savings for the health system because this will allow to make the diagnosis of melanoma in earlier stages and to save costs for unnecessary surgery. In this article we are going to review the technique.

Background/aims: Treatment of chronic delta hepatitis is long and difficult and better monitoring is needed., Methods: In this study, hepatitis delta virus (HDV) RNA, hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA were retrospectively quantified in 53 patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis delta. Twenty-one had received 28 courses of 3-5 MU interferon-alpha2b (IFN-alpha2b) thrice weekly for a median of 12.6 months (interquartile range [IQR]: 7.3-31.6), five had received eight courses of 100 mg lamivudine (LAM) daily for 23.6 months (IQR: 8.4-61.5) and 27 were untreated. The controls were 54 untreated, randomly selected, HBeAg-negative chronic hepatitis B patients without delta infection. Quantification of serum HDV RNA, HBsAg and HBV DNA were performed at baseline, during and at the end of treatment and end of follow up., Results: Untreated patients had significantly higher median HBsAg levels than controls (5,872 vs 3,501 IU/ml; P = 0.046), but lower median HBV DNA levels (2.933 vs 6.459 log10 copies/ml; P < 0.001). Median baseline HDV RNA (6.374 log10 copies/ml) was similar in IFN-alpha2b-treated, LAM-treated and untreated patients. At the end of treatment, IFN-alpha2b significantly suppressed in paired measurements HDV RNA (P = 0.012) and HBsAg (P = 0.043), but LAM was inefficient. In IFN-alpha2b-treated patients, HDV RNA became undetectable in five patients within a median of 30 months (IQR: 8-90), followed by a slower decrease in HBsAg., Conclusions: In untreated chronic delta hepatitis, suppressed HBV replication is associated with significantly increased HBsAg serum levels. IFN-alpha2b significantly suppresses both HDV RNA and HBsAg, but LAM has no effect. Long-term IFN-alpha2b treatment (IQR: 1.5-5.0 years) appears necessary for undetectable serum HDV RNA and further treatment is required for HBsAg loss. Monitoring of HDV RNA and HBsAg serum levels in patients with chronic delta hepatitis provides insight during treatment.

Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected by hepatitis B virus. Interferon alpha at high doses, although poorly efficient, is the only treatment reported to provide some benefit in chronic hepatitis delta. Pegylated interferon alpha (PEG-IFN) has not yet been evaluated. Treatment is usually monitored by the qualitative detection of HDV-RNA in serum. In this study, safety and efficacy of PEG-IFN were assessed in chronic hepatitis delta, and serum HDV-RNA kinetics were determined using quantitative RT-PCR. Fourteen patients with chronic hepatitis delta received subcutaneous PEG-IFN alpha-2b during 12 months (1.5 microg/kg per week). Serum HDV-RNA was quantified at initiation and during the course of therapy, and during the posttreatment follow-up period, which ranged from 6 to 42 months (median 16 months). PEG-IFN alpha-2b was well tolerated, inducing no serious adverse effect. Sustained biochemical response was obtained in 8 patients (57%). At the end of treatment, 8 patients (57%) had achieved virological response (undetectable HDV-RNA). Sustained virological response throughout the posttreatment follow-up period was observed in 6 patients (43%). HDV-RNA kinetics were predictive of the response: after 3 months of PEG-IFN, HDV-RNA levels were significantly lower in the responders than in the nonresponders group (P=.018). After 6 months of therapy, a negative HDV-RNA was predictive of sustained response (P=.021). In conclusion, this preliminary study indicates that PEG-IFN alpha-2b is safe and efficient for treatment of chronic hepatitis delta. The follow-up of HDV-RNA levels during therapy, which allows the differentiation of various profiles of virological responses, improves treatment monitoring.

Hepatitis delta virus is the only representative of the Deltavirus genus, which consists of 7 differentiated major clades. In this study, an eighth clade was identified from 3 distinct strains. Deltavirus genetic variability should be considered for diagnostic purposes. Clinical consequences of the diversity have yet to be evaluated.

The overall increase in relative survival rates of adult and childhood cancer patients is related to improved chemotherapy and radiotherapy treatment protocols. However, this success is tempered by gonadotoxic effects and uterine damage. The reduced fertility and possible premature menopause can have a profound impact on patients self-esteem and quality of life. Consequently, preservation of fertility or treatments to protect the ovarian function, become a more relevant issue. Advances in assisted reproduction techniques have focused on the following: embryo cryopreservation which is restricted by time and constrains such as, sexual maturity and the presence of a stable partner. Cryopreservation of immature ovarian tissue, the most promising option, with restoration of natural fertility following auto-transplantation or maturation of oocytes in vitro in combination with assisted reproduction.

Hepatitis delta virus (HDV), in association with hepatitis B virus, is responsible for severe acute and chronic hepatitis. Treatment of the infection relies on the long-term administration of high doses of alpha interferon (IFN), and the treatment efficiency is monitored by the detection of anti-HDV immunoglobulin M and HDV genome in serum. Like the case for other chronic viral infections, HDV genome quantification in serum should be useful for the follow-up of infected patients. The aims of this study were to develop a quantitative assay for the detection of any type of HDV in serum and to evaluate the benefit of HDV RNA quantification for the follow-up of chronically infected patients receiving IFN. A real-time reverse transcription-PCR assay was developed to quantify the HDV RNA load in serum. Its efficacy was evaluated with 160 serum samples, 76 of which were collected from 11 chronically infected patients who were treated with pegylated IFN. The assay was sensitive (100 copies/ml of serum) and efficient for all HDV types, including type 3 and the recently described types 5, 6, and 7. The viral load determinations for treated patients allowed us to identify different profiles of virological responses to IFN therapy with more accuracy than that attainable with the qualitative approach. In conclusion, we have developed a quantitative HDV RNA assay for serum which is adapted to the follow-up of antiviral treatment for patients infected with any HDV type. The assay will help us to understand the natural history of HDV infection and to define guidelines for the management of chronic delta hepatitis.

Vitiligo is a cutaneous pigmentary disorder characterized by the loss of melanocytes. An autoimmune mechanism is strongly suspected to be involved in this affection given that it is frequently associated with autoimmune hormonal disorders, and because antibodies directed against melanocytic antigens are found in the serum of patients with vitiligo. We examined the role of cellular immunity in melanoma-associated vitiligo by expanding infiltrating lymphocytes from fresh biopsy specimens of vitiligo patches in melanoma patients. The vitiligo-infiltrating lymphocytes were almost exclusively T lymphocytes, and most were CD8(+). Following in vitro expansion, vitiligo-infiltrating lymphocytes remained predominantly CD8(+) and expressed the cutaneous homing receptor CLA. Furthermore, vitiligo-infiltrating lymphocytes had a clonal or oligoclonal T cell receptor profile, possibly reflecting specific antigenic stimulation. Finally, vitiligo- infiltrating lymphocytes specifically recognized differentiation antigens shared by normal melanocytes and melanoma cells. This direct demonstration of CD8(+) T cell involvement in vitiligo suggests that, in melanoma patients, vitiligo may be a visible effect of a spontaneous antitumoral immune response.

The design of heteroclytic antigens with high MHC binding capacity is of particular interest to overcome the weak immunogenicity of peptide epitopes derived from tissue antigens expressed by tumors. In the present study, double-substituted peptide analogues of the tumor-associated antigen MART-1(27-35) incorporating a substitution at a primary anchor residue and a beta-amino acid residue at different positions in the sequence were synthesized and evaluated for binding to the human histocompatibility class I molecule HLA-A2 and for recognition by tumor-infiltrating lymphocytes. Interestingly, by combining a Leu for Ala substitution at P2 (which alone is deleterious for antigenic activity) with a beta-amino acid substitution at a putative TCR contact residue, recognition by tumor-infiltrating lymphocytes was partially restored. The analogue [Leu(28),beta-HIle(30)]MART-1(27-35) displays both a higher affinity to HLA-A2 and a more prolonged complex stability compared to [Leu(28)]MART-1(27-35). Overall, these results suggest that double-substitution strategies and beta-amino acid replacements at putative TCR contact residues might prove useful for the design of epitope mimics with high MHC binding capacity.

Replication-defective adenoviruses are arousing growing interest as both gene therapy and vaccine vectors. In a phase I clinical trial designed to evaluate the feasibility and tolerance of recombinant adenovirus (rAd)mediated gene transfer, we previously demonstrated that a single intratumoral injection of 10(9) PFU of rAd encoding the beta-galactosidase protein (Ad-beta-Gal) induced strong short-term (1-3 months) humoral, helper (Th1 type) and cytotoxic T cell responses specific for the transgene product in patients with advanced lung cancer. The purpose of the present study was to evaluate the persistence of long-lasting immunity to the transgene protein and in parallel, to assess patient immunocompetence revealed by responses to recall antigens (tetanus toxoid, purified protein derivative), viral pathogens (Epstein-Barr virus, influenza virus), and allogeneic antigens in mixed lymphocytic reactions. The beta-Gal-specific proliferative response declined rapidly in patients with progressive disease, as did responses to the other antigens. In contrast, a long-lasting proliferative response to beta-gal was maintained in an immunocompetent patient in complete remission 2 years after an injection of 108 PFU of Ad-beta-Gal. Anti-beta-Gal humoral (IgG and IgA) responses persisted notably, as did responses to TT and poliomyelytic antigens. While T cell effector cytotoxic responses specific for the viral peptides plummeted, the frequency of anti-beta-Gal CTL precursors remained particularly high, thus attesting to major immunization. Despite the impact of both advanced disease and chemotherapy on immunocompetence, we show the long-term persistence of immunity to the transgene protein vectorized by rAd.

A human immunodeficiency virus (HIV)-negative patient with no risk factor experienced HIV type 1 (HIV-1) primary infection 4 weeks after being hospitalized for surgery. Among the medical staff, only two night shift nurses were identified as HIV-1 seropositive. No exposure to blood was evidenced. To test the hypothesis of a possible nurse-to-patient transmission, phylogenetic analyses were conducted using two HIV-1 genomic regions (pol reverse transcriptase [RT] and env C2C4), each compared with reference strains and large local control sets (57 RT and 41 C2C4 local controls). Extensive analyses using multiple methodologies allowed us to test the robustness of phylogeny inference and to assess transmission hypotheses. Results allow us to unambiguously exclude one HIV-positive nurse and strongly suggest the other HIV-positive nurse as the source of infection of the patient.

Different parameters likely to influence the survival of bovine oocytes after a vitrification procedure were evaluated: oocyte meiotic stage, cycloheximide treatment at the beginning or the end of maturation, and three vitrification procedures using conventional straws, open pulled straws (OPS), or microdrops. For each procedure a mixture of cryoprotectants (25% ethylene glycol and 25% glycerol) was used. After the oocytes were warmed and subjected to in vitro maturation and fertilization, the number that developed into blastocysts was determined. Results show that cryoprotectant exposure reduced embryo development and that cycloheximide treatment had no beneficial effect on oocytes vitrified in conventional straws. Among the three vitrification procedures, only the OPS method yielded blastocysts (approximately 3% of vitrified oocytes) irrespective of their initial meiotic stage. This result highlights the major influence of the cooling rate in an oocyte vitrification protocol., (Copyright 1999 Academic Press.)

Nonclassical MHC class I HLA-G antigen expression is tissue specific and is thought to play a role in tolerance of the semiallogeneic fetus by the maternal immune system. Ectopic expression of HLA-G by tumor cells provides them with an additional mechanism of escape from immunosurveillance by host cytotoxic effector mechanisms. The aim of this study was to assess the expression of nonclassical HLA-G antigens in ex vivo human melanoma biopsies. HLA-G mRNA levels corresponding to both membrane-bound and soluble protein isoforms were analyzed in tumor specimens obtained from primary or metastatic melanomas of 23 patients. High levels of HLA-G transcription were detected in tumor specimens in 5 of 23 patients and found to be comparable in both lymph node and skin metastases. HLA-G mRNA transcript levels at tumor sites in 18 of these patients were compared with those in samples of their own healthy skin and were higher in the tumor tissue in 12 patients. Differential expression of mRNA transcripts corresponding to soluble and membrane-bound HLA-G was also observed in some tumor biopsies. HLA-G protein expression was detected in tumors that exhibited high levels of HLA-G transcription by immunofluorescence of frozen sections and Western blot analysis of both tumor and healthy skin biopsies, using anti-HLA-G-specific monoclonal antibodies. This work provides evidence that HLA-G gene transcription and protein expression can be up-regulated ex vivo in melanoma. Our finding that several of the tumors studied expressed high levels of HLA-G provides additional clues as to how a tumor can be selected in vivo to escape from cytotoxic antitumor responses, constituting a new parameter to be considered in the design of therapeutic approaches aimed at enhancing antitumor immune responses.

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

This study investigates the generation of primary melanoma cell-specific cytotoxic T lymphocytes (CTLs) in vitro. Induction of peptide-specific CTLs from unfractionated naive peripheral blood mononuclear cells from HLA-A2 healthy donors was assessed using 2 recently described 9-mer epitopes from the melanoma tumor antigen Melan-A/MART-1. The need for help from CD4+ T lymphocytes for the long-lasting induction of CTLs and the capacity of the peptide-induced CTL lines to recognize many melanoma cells were evaluated. CTL lines were obtained reproducibly when CD4+ T-lymphocyte help was provided during the primary stimulation either in an autologous way, in the case of tetanus toxoid antigen (TT) responder donors, or with allogeneic TT-activated T-helper cells, separated by an insert well, in the case of tetanus toxoid non-responder donors. We also investigated helper T-cell-derived factors that are produced by TT-activated lymphocytes. Our results strongly suggest that a complex network of cytokines like interleukin-2 (IL-2), interferon-gamma, IL-6 and IL-1 exerts stimulatory effects for the initiation process of CTLs. In contrast, cytokine-like IL-4 might inhibit generation of cytolytic activity if provided by TT-activated T cells at early stages of induction. Our approach can be used to generate CTLs of a desired specificity for clinical use in adoptive immunotherapy protocols.

Introduction: Because of the potential hepatotoxicity of retinoids, prescription of isotretinoin is always very carefully made in healthy subjects, and prohibited in case of concomitant hepatopathy. Gilbert's syndrome consists of chronic, mild, unconjugated hyperbilirubinemia. In this syndrome, isotretinoin has been reported twice to be perfectly tolerated, and once even beneficial. We report here a new case of good tolerance and even improvement of a Gilbert's syndrome during isotretinoin therapy., Case Report: A 17-year-old man with Gilbert's syndrome presented with a nodulocystic acne. Topical agents had been inefficient, and cyclines bad tolerated. Thus isotretinoin has been gradually introduced, with a regular monitoring of the liver function. We observed a steady decrease of the bilirubinemia during the course of isotretinoin, and then a reappearance of hyperbilirubinemia as soon as posology was diminished and particularly after completion of isotretinoin therapy., Discussion: A review of the literature finds only very few cases of hepatic injuries caused by isotretinoin, contrary to etretinate. Safety of isotretinoin in Gilbert's syndrome was first observed in 1984, but its beneficial effects have only recently been described by Wang et al., and we report here a similar case. Pharmacological mechanisms remain hypothetic. However, considering the prevalence of Gilbert's syndrome and its usual first expression during postpubertal period, it seems to us interesting for therapeutic practice to know that isotretinoin is not less safe in these patients.

To study the antiviral efficacy of high doses of alpha 2b interferon (alpha 2b-IFN) for chronic hepatitis D treatment, we used polymerase-chain-reaction(PCR)-based semi-quantitative detection of HDV RNA. The semi-quantification method used was based on the appearance of a positive amplification signal as a function of the number of PCR cycles. By amplifying dilutions (10(-1)-10(-8)) of an HDV-positive woodchuck liver RNA, we confirmed that exponential amplification efficacy occurred at between 20 and 30 cycles. Positive signals were observed from dilution 10(-2) (gel electrophoresis after 20 cycles of PCR) to dilution 10(-7) (hybridization after 30 cycles of PCR). To characterize the HDV RNA level in sera of 8 patients treated with alpha 2b-IFN (10 MU/3 times a week) for 1 year, we extracted RNA from serum samples taken every 6 months. All samples were amplified in parallel for 20 and 30 PCR cycles. Analysis of HDV cDNA after ethidium bromide/agarose gel electrophoresis and after molecular hybridization (100 times more sensitive than gel analysis), enabled us to grade the signals observed from negative to positive as 1+, 2+, 3+ and 4+, with all results being positive. Three types of evolution of HDV viraemia were evidenced among the 8 treated patients. HDV replication continued to occur at a high level at the 6th and 12th month in 2 patient sera. For 2 other patients, an HDV RNA decrease or disappearance was evidenced in the serum at the 6th month; however, viral replication recurred at a higher level at the 12th month.(ABSTRACT TRUNCATED AT 250 WORDS)

The recent development of co-culture technique has permitted to cryopreserve human embryos at the blastocyst stage. The aim of the present study was to compare pregnancy rates after transfers of embryos frozen at an early embryo stage (2-8 cell stage, group A; n = 175) with others frozen at blastocyst stage (group B; n = 98). Our results demonstrated that the frozen/thawed blastocyst survival is approximately 100%. In comparison the early embryos survival rate was 59% (P < 0.05). Moreover, pregnancy rates/embryo transfer in group A (16.4%) were significantly improved by comparison to those observed with early embryos (6.2%) (P < 0.05). In conclusion, our results demonstrate that embryo frozen/thawed at the blastocyst stage can be useful for improving pregnancy rates after cryopreservation.