Lentivirus-transduced human monocyte-derived dendritic cells efficiently stimulate antigen-specific cytotoxic T lymphocytes.

Dendritic cells (DCs) are professional antigen-presenting cells that are highly effective adjuvants for immunizing against pathogens and tumor antigens. The potential merit of genetic approaches to loading DCs with antigens is to express high and sustained levels of proteins that can be subsequently processed and presented to T lymphocytes. Replication-defective oncoretroviruses are able to efficiently transduce CD34(+) progenitor-derived DCs but not monocyte-derived DCs. Here, it is shown that efficient gene transfer is obtained using a human immunodeficiency virus-1-derived lentiviral vector deleted of all structural and accessory genes. Infection of immature DCs with the lentiviral vector at a multiplicity of infection of 20 resulted in stable gene expression in 30% to 40% of the matured DCs. Proviral DNA was detectable by Alu polymerase chain reaction for the lentiviral but not the oncoretroviral vector. Most importantly, it is demonstrated that lentivirus-transduced DCs were fully functional and effectively activated autologous HLA A2.1(+) peripheral blood cytotoxic T lymphocytes (CTLs). DCs expressing lentiviral vector-encoded Flu peptide were at least as efficient as DCs pulsed with the same peptide in stimulating specific CTLs. The efficacy of the lentivirus-transduced DCs was further demonstrated by their ability to directly activate freshly harvested peripheral blood Flu-specific CTLs in the absence of CD4(+) T-cell help and exogenous cytokines. The availability of a stable gene delivery system based on a multiply attenuated lentivirus that does not encode any viral protein and that allows sustained antigen presentation by DCs derived from blood monocytes will be very useful for the biologic investigation of DCs and the improvement of immunotherapeutic strategies involving DCs.

Restoration of alloreactivity of melanoma by transduction with B7.1.

Melanoma cells are unusual because, unlike most epithelial tumors, constitutive expression of human leukocyte antigen (HLA) class II molecules is common. To elucidate the role of HLA class II expression in the immunopathogenesis of melanoma, the authors compared HLA class II+ melanoma cells to autologous B cells with respect to their ability to stimulate primary (naïve) histoincompatible lymphocytes and T-cell clones (antigen experienced). Using primary lymphocytes (peripheral blood lymphocytes [PBLs]), melanoma cells were nonstimulatory when compared to autologous B cells. To determine whether this was caused by defective antigen processing, the authors used alloreactive T-cell clones, which require alloantigen presentation by a histocompatible stimulator cell but not costimulation. Melanoma cells stimulated the alloreactive T-cell clones in two of three clones tested, indicating that they processed and presented alloantigen. To determine whether the failure of melanoma cells to stimulate primary lymphocytes was caused by their inability to costimulate the T cells, the authors transduced the melanoma cells with B7.1 and achieved stable expression in more than 95% of the cells. The transduced cells were highly stimulatory, eliciting a 17- to 25-fold increase in proliferation by the peripheral blood lymphocytes compared with controls. Indeed, B7-expressing melanoma cells were more stimulatory than autologous B cells, which elicited an 11- to 15-fold increase compared with controls. These data indicate that melanoma cells fail to stimulate primary lymphocytes because they do not deliver costimulatory signals. Engineering HLA class II+ melanoma cells to express high levels of B7.1 may provide a way to elicit primary T-cell responses to melanoma-associated antigens.

Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells.

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) is a promising therapeutic approach for a number of diseases. To overcome the difficulty in generating specific CTLs, we established stable artificial antigen-presenting cells (AAPCs) that can be used to stimulate T cells of any patient of a given human leukocyte antigen (HLA) type. Mouse fibroblasts were retrovirally transduced with a single HLA-peptide complex along with the human accessory molecules B7.1, ICAM-1, and LFA-3. These AAPCs consistently elicit strong stimulation and expansion of HLA-restricted CTLs. Owing to the high efficiency of retrovirus-mediated gene transfer, stable AAPCs can be readily engineered for any HLA molecule and any specific peptide.

Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen.

The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel zeta chain fusion receptor specific for prostate-specific membrane antigen (PSMA) termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes.

Most tumor cells function poorly as antigen-presenting cells in part because they do not express costimulatory molecules. To provide costimulation to T lymphocytes that recognize tumor cells, we constructed a CD28-like receptor specific for GD2, a ganglioside overexpressed on the surface of neuroblastoma, small-cell lung carcinoma, melanoma, and other human tumors. Recognition of GD2 was provided by a single-chain antibody derived from the GD2-specific monoclonal antibody 3G6. We demonstrate that the chimeric receptor 3G6-CD28 provides CD28 signaling upon specific recognition of the GD2 antigen on tumor cells. Human primary T lymphocytes retrovirally transduced with 3G6-CD28 secrete interleukin 2, survive proapoptotic culture conditions, and selectively undergo clonal expansion in the presence of an antiidiotypic antibody specific for 3G6-CD28. Polyclonal CD8(+) lymphocytes expressing 3G6-CD28 are selectively expanded when cultured with cells expressing allogeneic major histocompatibility complex class I together with GD2. Primary T cells given such an antigen-dependent survival advantage should be very useful to augment immune responses against tumor cells.

Expression of a new M(r) 70-kDa dystrophin-related protein in the axon of peripheral nerves from Torpedo marmorata.

By comparison with localizations of dystrophin family products in rabbit peripheral nerves, we investigated the potential existence and distribution of similar products in peripheral nerves from Torpedo marmorata. In immunofluorescence studies, a specific set of monoclonal antibodies directed against dystrophin family proteins clearly stained a thin rim surrounding each Schwann cell-axon unit both in T. marmorata and rabbit peripheral nerves. In contrast when using the dystrophin/utrophin monoclonal H'3E7 antibody, we found a clear difference between rabbit and T. marmorata peripheral nerves according to fluorescent labeling detected within Torpedo nerve axons. Further differences were noted following western blot analyses of T. marmorata peripheral nerve extracts, highlighting the presence of a new and specific M(r) 70-kDa protein band belonging to the dystrophin family, which is localized within axons in addition to: (1) an M(r)400-kDa protein band detected with dystrophin/utrophin antibodies; and (2) an M(r) 116-kDa doublet protein band corresponding to Dp116 and Up116 isoforms. All of these products, detected according to the specificities of the monoclonal antibodies used, are discussed in terms of their potential identities as short and long dystrophin or utrophin mammalian products.

Identification of novel germline hMLH1 mutations including a 22 kb Alu-mediated deletion in patients with familial colorectal cancer.

We analyzed the hMLH1 gene in 17 unrelated families with putative hereditary nonpolyposis colorectal cancer. The complete hMLH1 cDNA was amplified in one step, and after a second amplification, four overlapping segments were directly sequenced. We detected, in five families that did not meet the complete Amsterdam criteria, five alterations, including a double-base change resulting in a missense mutation (Lys-618-Ala), a splicing mutation affecting the intron 4 splice acceptor site, a 2-bp deletion at codon 726, a 7-bp deletion at codon 626, and a deletion of exons 13-16. The latter alteration was shown to result from a 22-kb genomic deletion due to a homologous recombination between Alu repeats located in introns 12 and 16. The detection of five germline hMLH1 mutations in five families that only partially fulfilled the Amsterdam criteria shows that these criteria do not allow the identification of all familial colorectal cancers due to mutations of the mismatch repair genes. The numerous Alu repeats present within the hMLH1 gene and the observation of large genomic deletions suggest that (a) Alu-mediated deletions might frequently be involved in hMLH1 inactivation, and (b) reverse transcription-PCR analysis, which allows the amplification of the entire coding region of the hMLH1 gene in one step, might be the most appropriate method for the detection of hMLH1 alterations.

Presence of long and short dystrophin and/or utrophin products in Torpedo marmorata peripheral nerves.

Peripheral nerves from rabbit and Torpedo marmorata were comparatively analyzed for the presence of short dystrophin products. Western blot analyses of Torpedo marmorata peripheral nerve extracts revealed the existence of three proteins belonging to the dystrophin family: a M(r) 400 kDa protein band detected with dystrophin/utrophin, dystrophin-specific and Torpedo utrophin-specific antibodies, a molecule identified as Dp116 and, for the first time at the protein level, a new protein probably corresponding to Up116. All of these products were carefully identified according to the specificities of the monoclonal antibodies used. In immunofluorescence studies, clear staining of the thin rim surrounding each Schwann cell-axon unit was observed in both Torpedo marmorata and rabbit peripheral nerves, showing colocalization of all of these molecules. Their potential functions were discussed in comparison to similar products found in rabbit peripheral nerves.

[Acute necrotizing cutaneous streptococcal infection following bites or scratch by dog or cat]. / Streptococcies cutanées aiguës nécrosantes après morsure et griffure de chat et de chien.

INTRODUCTION: Domestic animal bites or scratches are quite frequent. Among banal bacteria isolated from infected bites or scratches, group A streptococcus seems to be frequently associated with severe infections. CASE REPORTS: Three cases of acute necrotizing cutaneous streptococcal infections, following cat or dog bite or scratch are reported. Twice, group A streptococcus was isolated from cutaneous swabs. In the third case, previous antibiotic therapy had sterilised bacteriological samples. Diagnosis was ascertained on the basis of clinical presentation and significant antistreptococcal antibodies elevation. Skin necrosis around the inoculation area was observed in the 3 cases. Cicatrisation required an average of two months under appropriate treatment. DISCUSSION: An evolution towards cutaneous necrosis localized to the initially injured area is common to these three cases. This peculiar evolution is worth to be known in order to choose an effective anti streptococcal antibiotherapy whenever domestic animals bites and scratches are to be treated.

Re-evaluation of the distributions of dystrophin and utrophin in sciatic nerve.

Differential expression of proteins belonging to the dystrophin family was analysed in peripheral nerves. In agreement with previous reports, no full-size dystrophin was detectable, only Dp116, one of the short dystrophin products of the Duchenne muscular dystrophy (DMD) gene. We used specific monoclonal antibodies to fully investigate the presence of utrophin, a dystrophin homologue encoded by a gene located on chromosome 6q24. Evidence is presented here of the presence of two potential isoforms of full-length utrophin in different nerve structures, which may differ by alternative splicing of the 3'-terminal part of the utrophin gene according to the specificities of the monoclonal antiobodies used. One full-length utrophin was co-localized with Dp116 in the sheath around each separate Schwann cell-axon unit, but the other utrophin isoform was found to be perineurium-specific. We also highlighted a potential 80 kDa utrophin-related protein. The utrophin distribution in peripheral nerves was re-evaluated and utrophin isoforms were detected at the protein level. This preliminary indication will require more concrete molecular evidence to confirm the presence of these two utrophin isoforms as well as the potential 80 kDa utrophin isoform, but the results strongly suggest that each isoform must have a specialized role and function within each specific nervous structure.

"In situ" characterization of GnRH receptors: use of two radioimagers and comparison with quantitative autoradiography.

New radioimagers, the HRRI (high resolution radioimager) and the Phosphorimager (phosphor screen : PS), apt to display more ample linear dose-response scale than radio-sensitive films, were tested in comparison with quantitative autoradiography (QA). GnRH receptor saturation experiments were achieved on tissue sections (rat pituitary, rat brain, human ovary) with a iodinate GnRH agonist (125I-[D-Ala6,Des-Gly10]-LH-RH Ethylamide) for determination of affinity constant (Kd). In rat pituitary, comparable results were obtained with the 3 methods (Kd: 0.4 to 0.6 nM). Discrepancies occurred in the hippocampus and in the granulosa cell layer of the preovulatory follicle, due to low resolutive (PS) or short linear dose-response (films) performances. In the hippocampus GnRH receptor affinity was under-estimated with PS (Kd: 2.3 vs 0.5 and 0.6 nM for QA and HRRI respectively). In the follicular granulosa cell layer it was over-estimated by QA (0.5 vs 50 nM for the HRRI), while PS did not allow resolution of this thin cell layer. In conclusion, the HRRI is a very powerful tool for the quantification of in situ radioligand binding (binding sites study and in situ hybridization) in very discrete areas.

Prolactin receptors in the rat hypothalamus: autoradiographic localization and characterization.

A precise mapping of prolactin (PRL) receptors in the rat brain has been achieved. Localization of binding sites for both 125I-human growth hormone (125I-hGH) and 125I-monoclonal anti-PRL receptor (125I-U5) was studied by in vitro autoradiography on brain sections in female rats (n = 7). The analysis of autoradiograms generated from 12 adjacent sections at 11 different brain levels (bregma 0.2 to -4.8 mm) revealed 9 distinctive localizations for 125I-hGH binding sites: preoptic suprachiasmatic nucleus, medial preoptic area, periventricular, supraoptic, paraventricular, arcuate and vetromedial nuclei and also the median eminence and the infundibulum. Specificity for PRL binding was assessed by competition experiment of 125I-hGH with unlabeled hGH and ovine PRL. Binding sites were similarly localized by 125I-U5 indicating the presence of PRL receptors moiety. The quantitative analysis with 0.6 nM 125I-hGH demonstrated maximal densities in the preoptic suprachiasmatic and arcuate nuclei and minimal densities in the median eminence and the infundibulum. Due to ample antero-posterior variations no significant changes were observed during the estrous cycle. Saturation analysis of binding in the arcuate nucleus indicated a single class of high affinity (Kd from 0.9 to 2.2 nM) receptors (Bmax from 34 to 44 fmol/mg of proteins). The present data provide the hypothalamic cartography of PRL receptors in the female rat brain and support all the physiological evidence for the existence of a direct action of PRL in the hypothalamus.

GnRH receptors in rat brain, pituitary and testis; modulation following surgical and gonadotropin-releasing hormone agonist-induced castration.

The regulation of rat gonadotropin-releasing hormone (GnRH) receptors in male rat pituitary, hippocampus and testis was studied, in vivo, under steady-state conditions during treatment with D-Trp6 GnRH (triptorelin, slow-release form, at 300 micrograms/kg/month). GnRH receptors were characterized on tissue sections by quantitative autoradiography using 125I-GnRHa as a tracer. Castrating doses of triptorelin strongly down-regulated pituitary GnRH receptors (50% of reduction after 8 h, 80% on days 1-30); in contrast, only a transient decrease (20% at 8 h) was observed in the hippocampus with a rapid return to control levels. Triptorelin induced a marked (2-fold) increase in GnRH receptors in testicular interstitial tissue during 5 days with a return to control value by day 20. Administration of a GnRH antagonist (BIM 21009, 1 mg/kg/24 h) induced a rapid reduction of pituitary and testicular receptors to undetectable levels at 24 h, while hippocampal receptors were strongly reduced only. This indicates that GnRH receptors with similar pharmacology are differently controlled in various tissues and that brain receptors are likely to be also regulated by GnRH agonists and antagonists.

[Post-traumatic cubitus varus in children. Apropos of 8 cases in African children]. / Le cubitus varus post-traumatique chez l'enfant. A propos de 8 cas chez l'enfant Africain.

The authors relate their experience with 8 cases of management of post-traumatic cubitus varus in the Africa child. In this series the mean varus angle, sense stricto, irrespective of the physiological valgus, was 24 degrees with extreme values ranging 10 degrees-45 degrees. Indication for surgery has always rested with the true varus value and with the degree of resulting esthetic and functional prejudice. Resection of an external bone wedge combined with synthesis using two crossed rods yielded good results, and rehabilitation was started as early as the first month after surgery.

GnRH receptors in human granulosa cells: anatomical localization and characterization by autoradiographic study.

The presence of receptors for GnRH in human ovary has been investigated by quantitative autoradiography. Simultaneous visualization and characterization of specific receptors on frozen sections were obtained on six pairs of human ovaries. Among them only one exhibited a large preovulatory follicle. This dominant follicle exhibited a specific and high affinity binding capacity for 125I-GnRHa exclusively localized on the granulosa cell layer. Analysis of saturation curve indicates a Kd value of 0.22 nM and Bmax of 9.6 fmol/mg protein. In contrast LH-hCG binding sites were present in all antral follicles. These data demonstrate for the first time the presence of high affinity GnRH receptors in human granulosa cells at a late stage of follicular maturation.

Characterization and distribution of receptors for gonadotropin-releasing hormone in the rat hippocampus.

Distribution and properties of receptors for gonadotropin-releasing hormone (GnRH) were analyzed in the brain of adult male rats. Binding of the iodinated GnRH agonist Des-Gly10-(D-Ala6)-GnRH ethylamide was studied in hippocampus and anterior pituitary using three convergent approaches: quantitative autoradiography on frozen tissue, binding to fresh slices, and binding to crude membrane preparations. In all cases, binding was specific, saturable, and time, pH, and temperature dependent. Quantitative autoradiography revealed that the density of binding sites was high in the stratum oriens and stratum radiatum of the CA1-CA4 regions of Ammon's horn. The pyramidal cell layer was faintly labelled. Binding was almost undetectable in the dentate gyrus. The highest density of sites (Bmax = 11.6 +/- 1.0 fmol/mg protein) was observed in the stratum radiatum of the CA3 region. Under the same conditions the value obtained for pituitary tissues was 20.7 +/- 2.8 fmol/mg protein. Analysis of saturation curves indicated only one class of high-affinity sites for the hippocampus (CA3; Kd = 0.28 +/- 0.03 nM) and for the pituitary (Kd = 0.29 +/- 0.08 nM). Both native GnRH and GnRH antagonist were potent competitors of binding. Fresh slices and membrane preparations from whole hippocampus confirmed these autoradiographic data and yielded affinity constants of 0.28 +/- 0.01 and 0.52 +/- 0.08 nM, respectively. In addition, a very high binding density was present in the amygdaloid complex, while binding was barely detectable in the hypothalamus. These results demonstrate that high densities of specific GnRH receptors are present in areas concerned with the regulation of behavioral functions.(ABSTRACT TRUNCATED AT 250 WORDS)