Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the ß5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

miR-491-5p-induced apoptosis in ovarian carcinoma depends on the direct inhibition of both BCL-XL and EGFR leading to BIM activation.

We sought to identify miRNAs that can efficiently induce apoptosis in ovarian cancer cells by overcoming BCL-X(L) and MCL1 anti-apoptotic activity, using combined computational and experimental approaches. We found that miR-491-5p efficiently induces apoptosis in IGROV1-R10 cells by directly inhibiting BCL-X(L) expression and by inducing BIM accumulation in its dephosphorylated form. This latter effect is due to direct targeting of epidermal growth factor receptor (EGFR) by miR-491-5p and consequent inhibition of downstream AKT and MAPK signalling pathways. Induction of apoptosis by miR-491-5p in this cell line is mimicked by a combination of EGFR inhibition together with a BH3-mimetic molecule. In contrast, SKOV3 cells treated with miR-491-5p maintain AKT and MAPK activity, do not induce BIM and do not undergo cell death despite BCL-XL and EGFR downregulation. In this cell line, sensitivity to miR-491-5p is restored by inhibition of both AKT and MAPK signalling pathways. Altogether, this work highlights the potential of miRNA functional studies to decipher cell signalling pathways or major regulatory hubs involved in cell survival to finally propose the rationale design of new strategies on the basis of pharmacological combinations.

[Antigenic peptides for peptide splicing in the proteosome]. / Peptides antigéniques produits par épissage peptidique dans le proteasome.

These past years, we focused our researches on the identification of novel, potential peptide targets for cancer immunotherapy. Amongst the peptides we identified, two are composed of fragments originally distant in the parental protein and are produced by a novel mechanism termed peptide splicing. The peptide splicing reaction takes place in the proteasome and occurs by transpeptidation. Here, we describe the discovery of this new mechanism of production of antigenic peptides.

Cognitive function and 99mTc-ECD brain SPECT are significantly correlated in patients with primary Sjogren syndrome: a case-control study.

OBJECTIVES: To assess subclinical central nervous system (CNS) involvement in primary Sjögren syndrome (pSS), by comparing standard brain MRI, in-depth neuropsychological testing and (99m)Tc-ECD brain single-photon emission computed tomography (SPECT) of patients with pSS with matched controls. METHODS: 10 women (<55 years old), with pSS defined using European-American criteria, presence of anti-SSA and/or anti-SSB antibodies and no history of neurological involvement were prospectively investigated, and compared with 10 age- and sex-matched controls. All subjects underwent, within 1 month, brain MRI, neuropsychological testing, including overall evaluation and focal cognitive function assessment, and (99m)Tc-ECD brain SPECT. RESULTS: (99m)Tc-ECD brain SPECT abnormalities were significantly more common in patients with pSS (10/10) than controls (2/10; p<0.05). Cognitive dysfunctions, mainly expressed as executive and visuospatial disorders, were also significantly more common in patients with pSS (8/10) than controls (0/10; p<0.01). Notably, between-group comparisons enabled a significant correlation to be established between neuropsychological assessment and (99m)Tc-ECD brain SPECT abnormalities in patients with pSS (r(s) = 0.49, p<0.01). MRI abnormalities in patients and controls did not differ significantly. CONCLUSIONS: Neuropsychological testing and (99m)Tc-ECD brain SPECT seem to be the most sensitive tools to detect subclinical CNS dysfunction in pSS. The strong correlation between cortical hypoperfusion in (99m)Tc-ECD brain SPECT and cognitive dysfunction suggests an organic aetiology of CNS dysfunction in pSS. These data should be confirmed in a larger study.

A peptide derived from melanocytic protein gp100 and presented by HLA-B35 is recognized by autologous cytolytic T lymphocytes on melanoma cells.

A panel of autologous cytolytic T lymphocyte (CTL) clones have been isolated from blood lymphocytes of a melanoma patient after in vitro stimulation with autologous tumor cells. We previously reported the molecular definition of three distinct antigens recognized by some of these CTL clones. We describe here, the identification of a fourth antigenic peptide expressed by this melanoma line and recognized by a CTL clone restricted by HLA-B*3503. The antigenic peptide, which is nine-amino acid long, has the sequence LPHSSSHWL and is derived from melanocyte differentiation antigen gp100. As HLA-B35 is one of the most frequent HLA-B alleles, being present in 20% of the Caucasian individuals, this peptide may be a good target for peptide-based immunotherapy of melanoma.

Interest of interferon alpha in systemic mastocytosis. The French experience and review of the literature.

Systemic mastocytosis (SM) are defined by an abnormal growth and accumulation of mast cells in bone marrow and/or other extracutaneous organs. There is currently no cure for this disease. Because of similarities and/or association of mastocytosis with myeloproliferative disorders, interferon alpha has been tested but with contradictory reported results. A first prospective multicenter phase II trial was then started in France. From 1994 to 1997, 20 adult patients with confirmed bone marrow involvement received interferon alpha-2b for at least 6 months, (from 1 million U per day up to 5 million U/m(2)/day). Thirteen patients who presented systemic and/or specific cutaneous manifestations, demonstrated objective responses: seven (35%) were partial, six (30%) minor but no complete response could be observed at the time of analysis. The bone marrow remained unchanged in 12/13. Thus, interferon should be offered to patients with severe systemic manifestations, who have not responded to symptomatic therapies, even in case of non-aggressive mastocytosis, with or without corticosteroids the first weeks. Long-term therapy should be offered to patients with initial positive response. To control more aggressive SM or mastocytosis associated with clonal hematologic non-mast cell lineage or leukaemia mast cell, other chemotherapeutic regimens should be proposed like Cladribine (2-chlorodeoxyadenosine, 2-CDA) or polychemotherapies including interferon as it is being tested in France in a new multicentric protocol, coordinated by the association AFIRMM, with interferon and oral cytarabine.

[Uterine fibroids embolization: results about 454 cases]. / Embolisation des fibromes utérins: résultats sur 454 cas.

OBJECTIVE: To determine the effectiveness of uterine arterial embolization (UAE) as primary treatment in the management of symptomatic leiomyomas. PATIENTS AND METHOD: UAE was performed on 454 patients (age range: 21-68) with menorrhagia, bulk-related symptoms or both, due to leiomyomas. The effectiveness of this therapy in the control of symptoms and reduction of uterine and leiomyoma volume was measured by clinical and imaging controls at 3, 6 and 9 months after the procedure. RESULTS: Four hundred and thirty-three patients were evaluated, and 42 failures were observed (9.6%). Six months after the procedure, 391 patients were symptom-free. Follow-up ultrasonic examination showed an average reduction of 55% in dominant myoma volume at 6 months, 70% at 1 year. Twenty-seven women became pregnant (30 pregnancies). Complications related to procedure, and requiring surgery, occurred in three cases. Principal complications are amenorrhoea and fibroid sloughs. Severe complications are rarely found. DISCUSSION AND CONCLUSION: UAE is an efficient therapy in the management of symptomatic myomas and proves to be a valid alternative to surgical procedure. The future of this mini-invasive and conservative technique appears to be a very promising one.

Lamivudine-zidovudine combination for prevention of maternal-infant transmission of HIV-1.

CONTEXT: Zidovudine reduces maternal-infant transmission of human immunodeficiency virus 1 (HIV-1) infection by two thirds. Combination antiretroviral therapies are potentially more effective prevention. OBJECTIVES: To assess the safety of perinatal lamivudine-zidovudine therapy, especially in children, and its effects on viral load, acquisition of drug resistance, and maternal-infant transmission of HIV-1 in a nonbreastfeeding population. DESIGN AND SETTING: The Agence Nationale de Recherches sur le SIDA (ANRS) 075 Study, an open-label, nonrandomized intervention trial conducted in the context of an ongoing observational cohort study in 48 sites in France. PATIENTS: A total of 445 HIV-1-infected pregnant women were enrolled as the study cohort from February 1997 to September 1998; controls consisted of 899 pregnant women who had received zidovudine monotherapy in May 1994 to February 1997 as standard care. INTERVENTION: The study cohort received lamivudine in addition to the standard Pediatric AIDS Clinical Trial Group 076 Study zidovudine prophylaxis regimen. Lamivudine was initiated in women at 32 weeks' gestation through delivery at 150 mg twice per day orally; children received lamivudine, 2 mg/kg twice per day for 6 weeks. MAIN OUTCOME MEASURES: HIV-1 infection status and tolerance of therapy in children through age 18 months; maternal plasma HIV-1 RNA levels through 6 weeks after delivery. RESULTS: The transmission rate in the study group was 1.6% (7/437; 95% confidence interval [CI], 0.7%-3.3%). In a multivariable analysis, transmission in the study group was 5-fold lower than in controls. In the study group, maternal plasma HIV-1 RNA level was less than 500 copies/mL at delivery in 74%; the median decrease was 1.24 (range, -1.63 to 3.40) log(10) copies/mL. The M184V lamivudine resistance mutation was detected at 6 weeks after delivery in specimens from 52 of 132 women. The most frequent serious adverse events in children were neutropenia and anemia, requiring blood transfusion in 9 children and premature treatment discontinuation in 19. Two uninfected children died at age 1 year from neurologic complications related to mitochondrial dysfunction. CONCLUSIONS: Lamivudine-zidovudine may be effective in preventing maternal-infant HIV transmission. However, severe adverse effects and emergence of resistance to lamivudine occurred. Thus, the role of this combination therapy in this setting is as yet unclear, and further research involving a variety of strategies is needed to definitively ascertain its utility for preventing maternal-infant HIV transmission.

Gallium-67 scintigraphy in macrophagic myofasciitis.

OBJECTIVE: To evaluate gallium-67 (67Ga) uptake and the value of 67Ga scintigraphy for diagnosis of macrophagic myofasciitis (MMF), a recently identified inflammatory myopathy. METHODS: Twelve consecutive patients with MMF confirmed by muscle biopsy, 10 with polymyositis, 10 with sarcoidosis, 8 with fibromyalgia, and 10 with lymphoma without muscle symptoms (serving as normal controls for muscle) were included. Patients received 1.8 MBq 67Ga per kg body weight by intravenous injection, and scintigraphy was performed with a 2-head gamma camera. The various views were acquired for the 3 main photopeaks of 67Ga 48 hours after infusion, and were analyzed in 2 blinded experiments by nuclear physicians. A semiquantitative scale was used to compare the uptake of 67Ga in the vascular soft tissue background with that in the muscles or joints of MMF patients, and with that in the normal controls. RESULTS: The MMF patients (4 men and 8 women, mean +/- SD age 47.8 +/- 8.7 years) had chronic myalgia (n = 11; predominantly in the lower limbs), asthenia (n = 10), arthralgia (n = 7), mild muscle weakness (n = 5), and high serum creatine kinase levels (n = 6). All MMF patients had significantly higher levels of 67Ga uptake in the muscle and para-articular areas than that recorded for the soft tissue background and for the controls. Muscle uptake was bilateral, symmetric, and homogeneous, and predominantly localized in the legs. No linear enhancement corresponding to fascias or synovial involvement was observed. In patients with polymyositis, symmetric, but heterogeneous, 67Ga uptake was observed in muscle, but not in the fascia. In patients with sarcoidosis, 67Ga uptake was nodular and heterogeneous in muscle, was not detected in the fascia, and was suggestive of synovial involvement in the joints. The uptake of 67Ga in fibromyalgic patients was similar to that in normal controls and to that in the soft tissue background. CONCLUSION: MMF is a new condition involving characteristic changes that can be detected by deltoid muscle biopsy. It usually manifests as a weakly specific, chronic arthromyalgic syndrome that predominates in the lower limbs. 67Ga scintigraphy is a noninvasive method that may make it easier to differentiate MMF from fibromyalgia and sarcoidosis.

Pregnancy after embolization of uterine myoma: report of 12 cases.

OBJECTIVE: To treat uterine myomas with embolization, to look for pregnancy-induced myoma recurrences, and to assess pregnancy course and outcome after embolization. DESIGN: Observational clinical study. SETTING: University of Paris VII hospital. PATIENT(S): Nine women had embolization for symptomatic myoma, with 12 pregnancies observed. INTERVENTION(S): Embolizations were highly selective and performed bilaterally through the uterine arteries with polyvinyl alcohol. MAIN OUTCOME MEASURE(S): Pregnant women were evaluated by physical and sonographic examinations. RESULT(S): Before embolization, the mean uterine volume was 450 cm(3), and in six patients polymyomas were present. The median age at embolization was 40 years; the median delay before pregnancy was 9 months; and the median age at first pregnancy outcome was 41 years. Five early miscarriages occurred. The seven other pregnancies were uneventful, although three premature births and one case of late toxemia occurred unrelated to previous embolization. Three women delivered vaginally and four by cesarean section. Neither myoma recurrence nor abnormality in uterine function was observed. CONCLUSION(S): The results of this first series of 12 pregnancies after myoma embolization are promising. If these preliminary results are confirmed, embolization could be a major breakthrough in the management of myoma and could replace conventional medical and surgical treatments.

[Arterial embolization of uterine myoma: results apropos of 286 cases]. / Embolisation artérielle des myomes utérins.

OBJECTIVE: Report outcome in non-surgical treatment of symptomatic uterine myomata by particulate arterial embolization. PATIENTS: and method: Two hundred eighty-six women aged 21 to 53 years with symptomatic uterine fibroids initially programmed for surgery were studied. The size and number of myomata were determined by pelvic ultrasound. After retrograde transfemoral introduction of a 4 French catheter, the left and right uterine arteries were successively catheterized. PVA particles were injected by free flow until devascularization. RESULTS: Two hundred sixty-two patients were evaluable. Complete resolution of symptoms was obtained in 245 cases. There were 17 failures. A marked reduction in the size of the myomata was observed (60% at six months). Hemorrhage disappeared in 80% of the cases immediately. Thirteen pregnancies were observed. No recurrence were observed. Complications were rare. CONCLUSION: Particulate embolization is a new minimally invasive treatment for uterine myomata which provides a alternative to hysterectomy and can replace myomectomy in young women.

Persistent mitochondrial dysfunction and perinatal exposure to antiretroviral nucleoside analogues.

BACKGROUND: Zidovudine is commonly administered during pregnancy to prevent mother-to-child HIV-1 transmission. We investigated mitochondrial toxic effects in children exposed to zidovudine in utero and after birth. METHODS: We analysed observations of a trial of tolerance of combined zidovudine and lamivudine and preliminary results of a continuing retrospective analysis of clinical and biological symptoms of mitochondrial dysfunction in children born to HIV-1-infected women in France. Mitochondrial dysfunction was studied by spectrophotometry and polarography of respiratory-chain complexes in various tissues. FINDINGS: Eight children had mitochondrial dysfunction. Five, of whom two died, presented with delayed neurological symptoms and three were symptom-free but had severe biological or neurological abnormalities. Four of these children had been exposed to combined zidovudine and lamivudine, and four to zidovudine alone. No child was infected with HIV-1. All children had abnormally low absolute or relative activities of respiratory-chain complexes I, IV, or both months or years after the end of antiretroviral treatment. No mutation currently associated with constitutional disease was detected in any patient. INTERPRETATION: Our findings support the hypothesis of a link between mitochondrial dysfunction and the perinatal administration of prophylactic nucleoside analogues. Current recommendations for zidovudine monotherapy should however be maintained. Further assessment of the toxic effects of these drugs is required.

Temporal 67gallium uptake is increased in temporal arteritis.

OBJECTIVE: We evaluated temporal 67gallium (Ga) uptake in temporal arteritis (TA) and the contribution of Ga scans to the diagnosis of TA. METHODS: Ga scans were performed prospectively in 19 patients with biopsy-proven TA and five TA patients with negative temporal artery biopsy. Controls were 18 elderly patients undergoing Ga scans for various inflammatory diseases. The temporal region of interest on head profiles was defined for comparison of uptake with a control parietal region of the same area. The Ga uptake ratio (GaUR) [(temporal region - parietal region)/parietal region] was evaluated for each temple by a computer and intra- and intergroup comparisons were made. RESULTS: GaUR was significantly higher in biopsy-proven TA patients (0.35+/-0.19) and biopsy-negative TA patients (0.31+/-0.03) than in controls (0.18+/-0.12) (P < 0.001), independently of recent temporal artery biopsy or short-duration steroid therapy. High GaUR (>0.4) had a 94% specificity and a 90% positive predictive value for TA diagnosis. After 6 months of steroid therapy, when patients were in remission, GaUR returned to baseline. CONCLUSION: Ga is specifically incorporated into the temporal area in TA patients which may be due to the granulomatous vasculitic process. Ga uptake ceases during remission. A high GaUR may contribute to TA diagnosis in temporal artery biopsy-negative patients and its role in the diagnosis of other localizations of the disease requires further evaluation.

67Ga scintigraphy in B-cell non-Hodgkin's lymphoma: correlation of 67Ga uptake with histology and transferrin receptor expression.

UNLABELLED: 67Ga scintigraphy is routinely used in the management of non-Hodgkin's lymphomas (NHLs), but the heterogeneity of 67Ga uptake in the different NHL histological subtypes has not been clearly explained. The transferrin receptors (TfR/CD71) play an important role in the mechanisms of 67Ga uptake by tumor cells. However, the relationship between the 67Ga uptake in NHL and the TfR/CD71 expression in lymphomatous cells remains to be defined. The aim of this study was to determine the intensity of 67Ga uptake in different histological subtypes of B-cell NHL (B-NHL) and to compare this uptake with the expression of TfR/CD71 on lymphomatous cells. METHODS: 67Ga scintigraphy of 47 patients having histologically proven lymphomas was investigated. 67Ga uptake was semiquantitatively evaluated in regions of interest and was reported as 67Ga uptake index (GaUI). In all cases, biopsies were reviewed for classification of NHL. The expression of TfR/CD71 was determined on frozen sections and was semiquantitatively evaluated. The relationships between GaUI, histology and TfR/CD71 were investigated. RESULTS: The values of GaUI were significantly related to the different histological subtypes analyzed (P = 0.0007) and to the presence of a large cells component, thus demonstrating that 67Ga uptake rose with the grade of lymphoma. Moreover, the values of GaUI and TfR/CD71 were closely related in the tested cases (P = 0.0059). CONCLUSION: There were three factors influencing 67Ga uptake in NHL: histology, TfR/CD71 expression and the presence of a large cells component. This justifies the usefulness of 67Ga scintigraphy in staging the TfR/CD71-positive lymphomas.

Central nervous system involvement in Sjögren's syndrome: evidence from neuropsychological testing and HMPAO-SPECT.

OBJECTIVE: To investigate the clinical, neuropsychological and imaging manifestations of Sjögren's syndrome (SS), a chronic auto-immune disease with peripheral and central nervous system (CNS) involvement. DESIGN/METHODS: Fourteen female patients suffering from confirmed SS underwent within 2 weeks: neurological examination, immunological staging, brain MRI, brain 99m Tc-HMPAO SPECT, psychological evaluation and in-depth neuropsychological testing. RESULTS: All patients showed neuropsychological abnormalities. The cognitive symptoms were of the same type in all patients, mostly frontal lobe syndrome and memory problems. The neuropsychological involvement was not associated with other kinds of CNS involvement or MRI abnormalities, but accurately reflected HMPAO imaging results. CONCLUSIONS: The results of this study indicate that cognitive evaluation is the most sensitive clinical test to diagnose CNS involvement in patients with SS, and that CNS involvement in SS seems to be more frequent when systematically assessed by neuropsychological tests.

Sarcoidosis activity: correlation of HRCT findings with those of 67Ga scanning, bronchoalveolar lavage, and serum angiotensin-converting enzyme assay.

PURPOSE: The objective of this study was to correlate the findings of sarcoidosis on high resolution CT (HRCT) with indexes of disease activity as measured with 67Ga scan, bronchoalveolar lavage (BAL), and serum angiotensin-converting enzyme (SACE) assay. METHOD: Twenty-nine patients with proven sarcoidosis underwent HRCT scan, 67Ga scan, BAL, and SACE assay within a 1 month period. The extent of parenchymal involvement by nodules, consolidation, ground-glass attenuation, and linear opacities was quantified to the nearest 10% of surface area affected on the CT examination. Whole-lung gallium uptake was quantified and the percentage of BAL-recovered lymphocytes (BAL-%LC) and SACE levels obtained by chart review. CT scores of disease extent were correlated with measured indexes of activity using the Spearman rank correlation coefficient. RESULTS: The mean extent of nodules, consolidation, ground-glass attenuation, and linear opacities on HRCT images was 15.1 +/- 16.6, 1.6 +/- 4.0, 17.5 +/- 25.4, and 7.6 +/- 9.6%, respectively. The extent of nodules and consolidation correlated with the intensity of lung gallium uptake (r = 0.46, p < 0.02), BAL-%LC (r = 0.50, p < 0.01), and SACE levels (r = 0.38, p < 0.05). No significant correlation was found between extent of ground-glass attenuation or linear opacities with any indexes of disease activity. CONCLUSION: On HRCT scan, nodules and consolidation in sarcoidosis reflect disease activity as measured by 67Ga scan, BAL, and SACE assay.

Mechanism involved in gallium-67 (Ga-67) uptake by human lymphoid cell lines.

The gallium-67 (Ga-67) is a radionuclide used for diagnostic imaging. It is known for its ability to accumulate in inflammatory lesions and tumors, especially in lymphomas. The intracellular distribution and the uptake mechanism of Ga-67 remains a subject of discussion. In this work, we studied the mechanism of Ga-67 uptake in 4 human lymphoid cell lines: 38658, Jurkat, DG75 and U715. Our results have pointed out the heterogeneity of Ga-67 uptake among these cell lines. Using flow cytometry analysis, we reported the expression of transferrin-receptor (TfR) in each lymphoid cell line, and found a positive correlation between TfR density and the Ga-67 uptake (r = 0.99) in lymphoid cells. The anti-TfR monoclonal antibody (MoAb) blocked significantly the Ga-67 uptake in all lymphoid cell lines indicating the Ga-67 uptake as a TfR-dependent mechanism. This mechanism has been saturated with different cold gallium concentrations. A total inhibition of Ga-67 uptake was obtained at a 10(-4) M cold gallium concentration. To conclude, we have shown that the Ga-67 uptake is TfR-dependent in lymphoid cells. Thus, the gallium penetrates in lymphoid cells by an active mechanism which can be saturated.