Th17 alloimmunity prevents neonatal establishment of lymphoid chimerism in IL-4-deprived mice.

Immune responses in newborn mice are known to be biased toward the helper type 2 phenotype. This may account for their propensity to develop tolerance. Herein, we evaluated the effects of IL-4 deprivation on CD4(+) T-cell activities elicited by neonatal exposure to allogeneic spleen cells. We showed that chimerism, Th2-type polarization and pathology, as well as skin allograft acceptance were inhibited in BALB/c mice immunized at birth with (A/J x BALB/c) F(1) spleen cells upon in vivo IL-4 neutralization. While IL-4 neutralization inhibited the development of Th2 cells in this model, it led to the accumulation of IL-17A, IL-17F, IL-22, IL-6 and RORγt mRNA in the spleen or graft tissues. Moreover, IL-4 deprivation led to the differentiation of donor-specific Th17 cells with a concomitant Th1 response characterized by IFN-γ production. The Th17-type response emerging in IL-4-deprived mice was found to mediate both intragraft neutrophil infiltration and the abrogation of B-cell chimerism. Neutralization of this Th17 response failed however to restore functional skin graft acceptance. Collectively, our observations indicate that the neonatal Th2 response opposes the development of Th17 cells, and that Th17 cells are responsible for controlling lymphoid chimerism in mice neonatally injected with semiallogeneic cells.

[Interest of immunohistochemic markers (Ki67, HMB45, p53) in risk analysis of congenital naevi of little and middle size]. / Intérêt des marqueurs immunohistochimiques (Ki67, HMB45, p53) dans l'analyse de risque des naevi congénitaux de petite et moyenne taille.

The risk to develop melanoma from small or medium size congenital naevus remain controversial. The main goal of the present study was to determine the interest of three immunohistochemical markers (Ki67, HMB45 and p53) in predicting malignant transformation of these congenital naevi and to see if a specific immunohistochemical profile of such transformed naevi can be identified. The markers (Ki67, HMB45 and p53) have been used retrospectively on sections of small or medium size congenital naevi (group NC, n = 15), of melanoma developed on small or medium size congenital naevi (group MNC, n = 15) and of melanoma developed on acquired naevi (group MNA, n = 15). The labelled cells have been counted in different cutaneous layers: junction, superficial dermal layer and deep dermal layer. No reactivity was observed for the three markers in group NC. The percentage of labelled cells was significantly different for the three markers between the group NC and the groups MNC and MNA. There was no difference between the groups MNC and MNA. In the groups MNC and MNA, a gradient in the percentage of labelled cells was observed between superficial and deep layers. These three markers do not differentiate melanoma developed from congenital naevi of small or medium size and melanoma developed from acquired naevi. Moreover, the results suggest that these three markers are useless in predicting the risk of malignant transformation of small or medium size congenital naevi.

CD8+ T-Cell depletion and rapamycin synergize with combined coreceptor/stimulation blockade to induce robust limb allograft tolerance in mice.

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti-CD154 mAb, nondepleting anti-CD4 combined to either depleting or nondepleting anti-CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long-term allograft survival in both combinations. Nevertheless, robust donor-specific tolerance, defined by the acceptance of a fresh donor-type skin allograft and simultaneous rejection of third-party grafts, required initial CD8(+) T-cell depletion. Mixed donor-recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL-2, IFN-gamma and TNF-alpha in MLC with donor antigen while significant alloreactivity persisted against third- party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor-recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8(+) T-cell depletion and costimulation/coreceptor blockade.

Skin biopsies in DC vaccines for stage III-IV melanoma patients: role of neutrophils?

Dendritic cell (DC) vaccines are used for the induction of anti-tumor T cell reaction in melanoma patients. DC are generated in vitro, pulsed with antigen and matured prior to injection. They are supposed to migrate to lymph nodes and to present the processed antigen to naive T cells allowing activation of tumor-specific lymphocytes. It has been suggested that intradermal injection allows a superior migration to the lymph node. Eight HLA-A2 positive patients with stage III or IV melanomas expressing NA 17 antigen were collected. They were included in a pilot trial of vaccination in which they received IL3/INFb DC presenting the NA17 A2 antigen. In each patient, a skin biopsy was performed at the injection site, 24 h after inoculation. The striking features of the biopsies were the presence of a perivascular CD3+/CD8+ T cell infiltrate with a slight population of CD4+ cells and the presence of a massive neutrophilic infiltrate associated with the injected DC still present, realizing a suppurative granuloma. The persistence of DC 24 h after the injection suggests that migration in the lymph node is not necessary for the induction of the immune response. The skin itself could be the location of a reaction starting with a massive recruitment of neutrophils.

Canine hyperplastic intraepidermal pustular and suprabasal acantholytic dermatosis with features of human pemphigus vegetans.

Pemphigus vegetans is a rare autoimmune blistering acantholytic dermatosis of humans that combines unusually hyperplastic and verrucous pustular skin lesions and mucosal erosions. We report herein the clinical, histopathologic, and immunologic findings in a dog whose lesions resembled, but were not identical to, those of human pemphigus vegetans. A 4-year-old male Greater Swiss Mountain Dog presented with multifocal cutaneous verrucous and crusted papules and pustules, as well as skin and mucosal erosions and ulcers. Microscopic lesions consisted of exophytic papillated epidermal hyperplasia, superficial and deep intraepidermal acantholytic neutrophilic and eosinophilic pustules, and suprabasal epidermal clefts leaving rounded basal keratinocytes at the bottom of the vesicles. Direct and indirect immunofluorescence revealed antikeratinocyte IgG autoantibodies. Immunoprecipitation immunoblotting and immunoabsorption experiments with recombinant canine desmogleins confirmed that autoantibodies recognized desmoglein-1. In this dog, clinical and histopathologic features resembled those of human pemphigus vegetans, while circulating autoantibodies against canine desmoglein-1 were solely identified. This antigen target is different from that of the human disease in which antidesmoglein-3 autoantibodies are detected most commonly.

Decreased immunoreactive maspin expression in intermediate thickness and thick primary melanoma lesions.

Maspin is a member of the serpin family of protease inhibitors. It is a 42 kDa cytoplasmic protein that is reported to have tumour suppressor activity. The loss of maspin gene expression is correlated with increased invasiveness and the risk of metastases in breast cancer. We studied maspin expression in primary melanoma lesions obtained from 76 patients. Immunostaining of 5 pm sections for maspin expression was obtained using the citrate antigen retrieval method. The extent of immunostaining was scored by recording the proportion of immunoreactive cells and the intensity of immunostaining. Our results demonstrated that maspin expression was down-regulated in intermediate thickness and thick melanoma lesions compared with thin lesions. These results suggest that loss of maspin expression might play a role in melanoma progression, invasion and metastatic dissemination. Further studies are needed to clarify the clinicopathological significance of maspin expression in melanoma.

Colorimetric analysis of pigmented skin lesions: a pilot study with the Visi-Chroma VC-100 device.

BACKGROUND: Definition of the colour of pigmented skin lesions (PSLs) with the naked eye remains subjective and may be influenced by lighting. This problem underlines the usefulness of instrumental assessments such as epiluminescence microscopy and colorimetric devices. OBJECTIVE AND METHODS: We describe here a new method of colour analysis of PSLs with the Visi-Chroma VC-100 device, which illuminates the surface of the skin with white light-emitting diodes (LEDs) and analyses the reflected light by a red-green-blue (RGB) charge-coupled device (CCD) colour camera. Twenty-one PSLs to be excised for cosmetic or medical reasons were analysed by this device with clinicopathological correlation. CONCLUSIONS: This method is feasible and might be useful to assess the colour of PSLs and allow comparisons for changes over time. Further studies are needed to determine the usefulness of this device in clinical practice.

High serum galectin-3 in advanced melanoma: preliminary results.

BACKGROUND: Galectin-3 (Gal-3) is a member of the family of beta-galactoside-binding mammalian lectins, and has been implicated in tumour invasion and metastatic process in vitro and in vivo. AIM: To determine whether an increase in serum Gal-3 production could be found in patients with advanced metastatic melanoma. METHODS: We collected 18 sera from patients with AJCC stage IV metastatic melanomas and 20 sera from healthy volunteers. Determination of Gal-3 was performed by ELISA, and in the group of patients with melanoma, these results were compared with the serum lactate dehydrogenase (LDH) and the C-reactive protein (CRP) concentrations. RESULTS: Gal-3 concentration was shown to be significantly higher in the group of patients with melanoma compared with healthy volunteers, and Gal-3 concentration was significantly correlated with both LDH and CRP in the melanoma group. We also selected four patients in the melanoma group for Gal-3 retrospective immunostaining analysis on cutaneous metastases. Two of these patients, who had a higher Gal-3 serum level, showed more intense staining and the other two patients, with a lower serum level of Gal-3, had moderate immunostaining, suggesting that at least part of serum Gal-3 might be produced by metastatic melanoma tissue. CONCLUSIONS: Gal-3 might play a role in melanoma progression and/or inflammation, and warrants further study.

Renal pelvis haemangioma demonstrated by MSCT urography with ureteral compression and 3D reconstruction.

A rare case of renal haemangioma of the renal pelvis is reported. The 40-year-old patient classically presented with recurrent hematuria. The small pyelic tumour was clearly delineated by msCT urography obtained during compression of the uretera with rubber balloons classically used during IVP In the absence of a definite histological diagnosis--pyeloscopy being unsuccessful--the patient underwent radical nephrectomy. The literature concerning renal haemangioma is briefly reviewed and the role of compression assisted msCT urography as a possible method for the detection of small pyelocaliceal tumours is emphasized.

Evaluation of extensive initial staging procedure in intermediate/high-risk melanoma patients.

INTRODUCTION: Early diagnosis and treatment of metastases have been shown to improve overall survival of melanoma patients. The purpose of this study was to evaluate the impact of extensive initial staging, including positron emission tomography (PET) scan on the management of melanoma patients. PATIENTS AND METHODS: Forty-three patients with intermediate/poor prognosis primary melanoma benefited from complementary excision and sentinel lymph node biopsy (SLB) after clinical and paraclinical staging (computed tomography, nuclear magnetic resonance and whole body fluorodeoxyglucose PET scan). RESULTS: No systemic metastases were demonstrated, while the SLB procedure emphasized the presence of regional lymph node metastases in 10 patients as suggested by the PET scan in four patients (sensitivity of the PET scan 40%). These 10 patients with early diagnosed lymph node involvement benefited from early surgery and were included in adjuvant treatment protocols. A secondary primary cancer was fortuitously diagnosed and treated early in two patients. CONCLUSIONS: The development of new adjuvant therapies and therapeutic procedures (specific and non-specific immunotherapy, gamma-knife radiosurgery, etc.) now raises the relevance of extensive staging in intermediate/poor prognosis melanoma patients. We confirm in our series that PET scan is not useful to detect micrometastasis and cannot replace SLB in initial regional staging. However, we show in our study that 12 of 43 patients were treated early or were included early in treatment protocols thanks to the extensive staging procedure. Nevertheless, it seems important to evaluate through larger prospective trials the real impact of these early diagnoses and new treatments on overall survival before defining new diagnostic and therapeutic guidelines.

In vitro evaluation of the marginal seal of four restoration materials on deciduous molars.

The aim of this study was to compare the marginal microleakage of Fuji II LC (A), composite resin Z250 (B), Fuji IX GP (C), and Dyract AP (F) in class V cavities and at the Fuji II LC/Z250 (D) and Fuji IX GP/composite resin Z250 (E) interfaces of an open sandwich technique on deciduous teeth. After thermocycling the mean marginal dye penetration at the enamel junction was 21.6 microns +/- 14.2 for group A; 83.6 microns +/- 32.3 for group B; 7.5 microns +/- 7.5 for group C; 38.7 microns +/- 27.5 for group D, and 0 micron for groups E and F. Mean dye penetration at the cementum junction was 37.1 +/- 20.2 (A); 123 +/- 42.1 (B); 28.7 +/- 17.1 (C); 0 (D); 14.4 +/- 14.4 (E); and 0 (F) microns. No leakage was seen at the junction between Fuji II LC and Z250 (0 micron), whereas a mean leakage of 184 microns between Fuji IX and Z250 was measured. In enamel the best seal was obtained with Dyract AP, but with differences at the limit of significance (P = 0.07). Sealing was significantly worse with Z250 (p = 0.03 versus Fuji II LC; p = 0.006 versus Fuji IX GP; and p = 0.003 versus Dyract AP). In cementum, the comparison between the grouped data Z250-Fuji II LC versus Fuji IX GP-Dyract AP was highly significant (p < 0.001), while there was no detectable difference between Z250 and Fuji II LC.

Spread of melanoma after lymphatic drainage: relaunching the debate.

Secondary lymphoedema of the leg can result in disruption of lymphatic vessels following lymph node surgery. Evidence supports the use of complex decongestive physiotherapy (CDP) in such cases, despite the possibility of tumour recurrence due to this therapy in cancer patients. We present the case of a 52-year-old woman who developed in-transit metastases and systemic evaluable disease one month after starting CDP for secondary lymphoedema of the leg.

BM-573, a dual thromboxane synthase inhibitor and thromboxane receptor antagonist, prevents pig myocardial infarction induced by coronary thrombosis.

The aim of this study was to characterize the effects of BM-573 [N-terbutyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl] urea], a novel dual thromboxane A2 receptor antagonist and thromboxane synthase inhibitor, on myocardial infarction induced by topical ferric chloride (FeCl3) application to the left anterior descending (LAD) coronary artery in anesthetized pigs. All control animals (n = 6) developed an occlusive thrombus in the LAD coronary artery. The mean infarct size, revealed by triphenyl tetrazolium chloride (TTC), and the area at risk, evidenced by Evans blue, corresponded to 35.3 +/- 2.2 and 36.9 +/- 2.1% of the left ventricular mass, respectively. In the BM-573-treated group (n = 6), a drug infusion (10 mg. kg-1. h-1) started 30 min before FeCl3 application and continued throughout the experimentation. Among the BM-573-treated group, four pigs did not develop coronary artery thrombus and their myocardium appeared healthy. Histopathological examination of FeCl3-injured coronary artery revealed an occlusive and adherent thrombus in control group, while pretreatment with BM-573 prevented thrombus formation. In infarcted zones, lack of desmin staining and muscle structure disorganization were obvious. Depletion of myocardial ATP content was observed in the myocardial necrotic region of the control group, but not in myocardial samples of BM-573-treated pigs that did not develop myocardial infarction. When BM-573 prevented LAD artery occlusion, the area under the curve of plasmatic troponin T was reduced by 77% over 6 h. These data suggest that BM-573 could be useful for the prevention of myocardial infarction.

Immunohistochemical profile of galectin-8 expression in benign and malignant tumors of epithelial, mesenchymatous and adipous origins, and of the nervous system.

This study aims to investigate whether the immunohistochemical expression of galectin-8 could be used as a diagnostic marker in tumor tissues of various histogenetic origins including specimens from epithelial (n=145), mesenchymatous (n=16), adipous (n=10) and central and peripheral nervous system (n=25) tissue, and 4 mesotheliomas. Immunohistochemical reactions were carried out with a polyclonal anti-galectin-8 antibody and histological slides from tissues derived from the files of the Laboratory of Anatomopathology of University Erasmus Hospital, Brussels. Formalin-fixed paraffin-embedded tissues of 45 normal cases as well as 41 benign and 114 malignant tumors were studied. Marked decreases in immunohistochemical galectin-8 expression were observed in colon (p=0.001), pancreas (p=0.007), liver (p=0.0008), skin (p=0.002) and larynx (p=0.02) tissue when comparing malignant tissue to normal tissue and/or benign tumors. The reverse relationship was observed for breast tissue (p=0.007). No statistically significant differences (p>0.05) were detected when comparing normal tissue and/or benign to malignant tumors in lung, bladder, kidney, prostate and stomach tissue. Significant galectin-8 expression was also measured in non-epithelial tissue including tumors of the central and peripheral nervous system as well as in skeletal muscle and mesotheliomas. Immunohistochemical monitoring of galectin-8 thus reveals an organ-type-dependent regulation of expression upon malignant transformation of various tissue types of epithelial origin. This observation will prompt further studies to delineate any relationship with prognosis.

Identification by quantitative chromatin pattern analysis of patients at risk for recurrence of superficial transitional bladder carcinoma.

PURPOSE: Based on the actual clinical outcomes of 132 fully documented patients with superficial transitional cell carcinoma of the bladder, we characterize the risk of recurrence and/or progression by computer assisted image microscopy applied to Feulgen stained nuclei. MATERIALS AND METHODS: Each tumor was characterized by the conventional grading and staging systems as well as by cytometry generated variables describing nuclear DNA content, nuclear morphometry and chromatin patterns. These data were submitted to discriminant analysis to establish a model distinguishing between 2 groups of patients. Group 1 included cases with remission for more than 60 months and group 2 cases presented with recurrence with or without progression within 12 months of transurethral bladder resection. This latter model was then validated by Kaplan-Meyer analysis of the full data set. RESULTS: As evidenced by Kaplan-Meier analysis, the discriminant factor generated by discriminant analysis of cytometry generated variables provided a cutoff value for distinguishing between low and high risks of recurrence (p <0.00001). In contrast, conventional grading and staging systems were not able to make such efficient distinction. CONCLUSIONS: These 2 groups can be used as references with which new cases can be compared to prognosticate disease behavior independently of histopathological grading and/or clinical staging.

Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi)

BACKGROUND: Chinese-herb nephropathy is a progressive form of renal fibrosis that develops in some patients who take weight-reducing pills containing Chinese herbs. Because of a manufacturing error, one of the herbs in these pills (Stephania tetrandra) was inadvertently replaced by Aristolochia fangchi, which is nephrotoxic and carcinogenic. METHODS: The diagnosis of a neoplastic lesion in the native urinary tract of a renal-transplant recipient who had Chinese-herb nephropathy prompted us to propose regular cystoscopic examinations and the prophylactic removal of the native kidneys and ureters in all our patients with end-stage Chinese-herb nephropathy who were being treated with either transplantation or dialysis. Surgical specimens were examined histologically and analyzed for the presence of DNA adducts formed by aristolochic acid. All prescriptions written for Chinese-herb weight-reducing compounds during the period of exposure (1990 to 1992) in these patients were obtained, and the cumulative doses were calculated. RESULTS: Among 39 patients who agreed to undergo prophylactic surgery, there were 18 cases of urothelial carcinoma (prevalence, 46 percent; 95 percent confidence interval, 29 to 62 percent): 17 cases of carcinoma of the ureter, renal pelvis, or both and 1 papillary bladder tumor. Nineteen of the remaining patients had mild-to-moderate urothelial dysplasia, and two had normal urothelium. All tissue samples analyzed contained aristolochic acid-related DNA adducts. The cumulative dose of aristolochia was a significant risk factor for urothelial carcinoma, with total doses of more than 200 g associated with a higher risk of urothelial carcinoma. CONCLUSIONS: The prevalence of urothelial carcinoma among patients with end-stage Chinese-herb nephropathy (caused by aristolochia species) is a high.

Characterization of steroid hormone sensitivity in human breast cancers maintained ex vivo under organotypical culture conditions.

PURPOSE: The methodology we propose combines the immunohistochemical determination of the oestrogen and progesterone receptors (ER and PgR) with the characterization of the oestradiol- and progesterone-induced influence on cell proliferation in breast cancers in order to characterize their steroid hormone sensitivity at both the "static" and "dynamic" level. METHODS: ER and PgR have been immunohistochemically quantified by means of computer-assisted microscopy. Cell proliferation has been determined by means of tritiated thymidine autoradiography in tumour samples maintained in vitro as organotypic cultures. A series of 14 patients was investigated. RESULTS: Of the 14 breast cancers under study, one with an unequivocally "very ER-rich"/"very PgR-rich" immunohistochemical phenotype totally failed to exhibit any modification in its cell proliferation level after both oestradiol and progesterone stimulation. Two cases definitively associated with an "ER-poor"/"PgR-poor" immunohistochemical phenotype nevertheless responded noticeably to the dynamic stimulation of their cell proliferation by oestradiol and progesterone. While our series of cases covers 14 patients only, it suffices to demonstrate the limits of ER and PgR determination in characterizing steroid hormone sensitivity in breast cancer. DISCUSSION: The present work therefore presents an in vitro approach to test growth regulation of human breast cancer by steroid hormones. The clinical value of the present approach should be further determined by showing that steroid hormone-induced modifications in cell proliferation level are actually associated with clinical response.

The importance of measuring the prostatic transition zone: an anatomical and radiological study.

OBJECTIVE: To assess the accuracy and reliability of measurements of the volume of the transition zone (TZ, representing the hypertrophied benign component) and whole prostate by TRUS in patients with BPH or cancer, by comparing the radiological with pathological findings after surgery. PATIENTS AND METHODS: The study comprised 36 patients with prostate cancer undergoing radical prostatectomy and 34 patients with symptomatic BPH treated using retropubic adenectomy. The weights of the radical prostatectomy specimens and of the enucleated adenomas were correlated with the corresponding volumes of the TZ and of the whole prostate, respectively, measured by TRUS using the prolate ellipsoid method. RESULTS: The mean (sd) TZ volume measured by TRUS was 36.9 (25.48) mL, whereas the weight of the enucleated specimen was 42.7 (33.58) g (correlation coefficient r=0.95, P<0.001). The TRUS estimate of the volume of the whole prostate was 29.2 (9.24) mL, while the radical prostatectomy specimens weighed 34.5 (10.76) g (r=0.78, P<0.001). The variability in the TZ volume estimate ranged from -17% to +18%, whereas the variability for whole prostate was -21% to +30% (P<0.05). CONCLUSIONS: Measurements of the TZ of the prostate by TRUS are more accurate than those for the whole prostate. An assessment of the TZ volume may be sufficiently reliable to be used in the clinical management of BPH and to detect prostate cancer using the prostate-specific antigen density of the TZ as a marker.