Direct intralymphatic injection of peptide vaccines enhances immunogenicity.

Research to enhance the efficiency of vaccines focuses mainly on improving either the adjuvant or the type and form of the antigen. This study evaluates the influence of the administration route on the efficiency of a peptide-based vaccine. Peptide vaccines are generally administered subcutaneously or intradermally, from where they must reach secondary lymphatic organs to induce an immune response. We analyzed the efficacy of peptide vaccines administered directly into a lymph node. Using a MHC class I-binding peptide from lymphocytic choriomeningitis virus, we found that intralymphatic injection enhanced immunogenicity by as much as 10(6) times when compared to subcutaneous and intradermal vaccination. Intralymphatic administration induced CD8 T cell responses with strong cytotoxic activity and IFN-gamma production that conferred long-term protection against viral infections and tumors. These results should have immediate implications for clinical immunotherapy of infectious disease and cancer.

Improved risk-benefit ratio for topical triamcinolone acetonide in Transfersome in comparison with equipotent cream and ointment: a randomized controlled trial.

BACKGROUND: Transfersome is a drug delivery technology based on highly deformable, ultraflexible lipid vesicles which penetrate the skin when applied non-occlusively. OBJECTIVES: To assess the advantages of this carrier-based formulation in humans, the efficacy and the atrophogenic potential of triamcinolone acetonide (TAC) in Transfersome was compared with commercially available TAC-containing cream and ointment. METHODS: Healthy volunteers were enrolled in double-blind, placebo-controlled clinical trials with random study medication assignment to the test areas. RESULTS: A 10-fold lower dose of TAC in Transfersome(R) (2.5 micro g cm-2) was bioequivalent to 25 micro g cm-2 TAC in conventional formulations as measured by erythema suppression (cream: P = 0.01, ointment: P < 0.001). A skin blanching assay revealed different kinetics of the formulations, with a delayed onset of action of the Transfersome and ointment preparations. Ultrasonic measurements revealed a significantly reduced atrophogenic potential. There was a 12.1% reduction in skin thickness given by TAC in Transfersome compared with a 21.1% reduction given by a bioequivalent dose in TAC cream after a 6-week treatment period (P = 0.007). CONCLUSIONS: Transfersome may significantly improve the risk-benefit ratio of topically applied glucocorticosteroids.

Intralymphatic immunization enhances DNA vaccination.

Although DNA vaccines have been shown to elicit potent immune responses in animal models, initial clinical trials in humans have been disappointing, highlighting a need to optimize their immunogenicity. Naked DNA vaccines are usually administered either i.m. or intradermally. The current study shows that immunization with naked DNA by direct injection into a peripheral lymph node enhances immunogenicity by 100- to 1,000-fold, inducing strong and biologically relevant CD8(+) cytotoxic T lymphocyte responses. Because injection directly into a lymph node is a rapid and easy procedure in humans, these results have important clinical implications for DNA vaccination.

[Ultrasound biomicroscopy and therapy of malignant glaucoma]. / Ultraschallbiomikroskopie und Therapie des malignen Glaukoms.

BACKGROUND: Malignant glaucoma is a rarely diagnosed condition though it has been known since over one hundred years and understood to be based on an ciliary blockage since thirty years. Now it is possible to visualise pathomechanism of ciliary block by ultrasoundbiomicroscopy. PATIENTS AND METHODS: Between January 1994 and November 1998 thirteen patients with ciliary block glaucoma had been observed. Four underwent ultrasoundbiomicroscopy. RESULTS: Ciliary block glaucoma is caused by obliteration of the posterior chamber. Ultrasoundbiomicroscopy showed, that in phakic eyes the lens, in pseudophakic eyes the capsule together with the anterior vitreous membrane and in aphakic eyes the vitreous alone are the blocking agents. Hyperopia, a narrow iridocorneal angle and ciliary sulcus as well as plateau iris configuration and a history of miotics are the predisposing risks for ciliary block glaucoma, especially after additional surgery such as cataract extraction, iridotomy, iridectomy and trabeculectomy. Clinical features are always a raised intraocular tension accompanied with a flattening of the anterior chamber, which are to be differentiated from an angle closure glaucoma. This is easy, if iridectomy, irido-capsulovitreotomy or pseudophakia are present and difficult in the very rare spontaneous cases. Cycloplegics and YAG-laser iridectomy may break the ciliary block, but the most preferable therapy is lensectomy (phakic eyes) and partial removing of the anterior vitreous and a peripheral sector of lens capsule combined with an iridectomy. This is easily performed with the vitrector via pars plana. CONCLUSIONS: Ultrasoundbiomicroscopy starts to confirm the theories on ciliary block glaucoma and allows to assess the different modes of treatment. The most successful treatment is lens extraction and partial vitreo-capsulo-iridectomy via pars plana.

Strongly reduced expression of the cell cycle inhibitor p27 in endometrial neoplasia.

In the present study we investigated the expression of the cell cycle inhibitor p27 in endometrial neoplasia using immunohistochemistry with a p27-specific antibody. Expression of p27 in endometrial carcinomas was compared with expression in the normal endometrium throughout the cycle. Normal endometrial cells showed strong nuclear expression of p27. Expression was present throughout the cycle and was stronger during the secretory phase. We found strongly reduced or abolished expression of p27 in endometrial carcinoma (85.3% of cases). The 41 tumours analysed were classified according to p27 staining intensity and percentage of positive cells into the following categories of p27 expression: negative/very low (56.0%); low (29.3%); moderate (14.7%) and high (0.0%). All the p27-positive tumours were well-differentiated endometrioid carcinomas of malignancy grade G1. Comparison with the p53 status showed that all tumours with strong p53 expression had low/negative p27 staining, while those that were positive for p27 had negative/low p53 staining. Reduced or absent p27 levels were also observed by Western blot analysis both in tumour samples and in HEC-1B endometrial adenocarcinoma cells. It thus seems that p27 expression is essential for the control of normal endometrial proliferation, and reduced or absent p27 expression may be an important step in endometrial carcinogenesis.

Dysregulated expression of CD66a (BGP, C-CAM), an adhesion molecule of the CEA family, in endometrial cancer.

CD66a (BGP, C-CAM) is an adhesion molecule of the carcinoembryonic antigen family that has been shown to be down-regulated in colorectal, prostate, and breast cancers. The purpose of the present study was to determine its expression pattern in the normal human endometrium and in endometrial neoplasia. For this purpose, we performed immunohistochemistry using the 4D1/C2 monoclonal antibody on a series of 24 normal endometrial samples and 47 endometrial carcinomas. Strong CD66a expression was observed in glandular and luminal epithelial cells of the normal endometrium with a consistent localization at the apical poles of these cells throughout the cycle. In late secretory (premenstrual) phase, loss of cellular polarity resulted in a membranous expression pattern in some glandular cells. In the analyzed tumor samples increasing areas with a complete loss of expression were observed with increasing malignancy grade. The apical expression pattern of the normal epithelium was changed to a membranous all-around pattern in 55% of the tumors, mostly in solid areas. This change correlated with malignancy grade and could be observed in 3 of 15 G1 tumors, 4 of 12 G2 tumors, 11 of 12 G3 tumors, and 8 of 8 serous-papillary carcinomas. Areas with membranous expression pattern could be observed along with areas with a normal apical expression pattern in lower grade carcinomas and with areas with complete loss of expression in high grade tumors. Northern blot analysis showed a loss of mRNA expression in tumor samples and HEC-1B endometrial adenocarcinoma cells. Loss of protein expression in the tumor samples was also observed by Western blot. In conclusion, CD66a protein expression is dysregulated in endometrial carcinomas, showing reduction or loss of expression with increasing malignancy grade and a change from the apical to a membranous localization.

Influence of urogenital infections on sperm functions.

Many studies have examined the impact of genital tract infections on male fertility; however, the effect of bacteriospermia on sperm quality is still controversial. Bacterial infections are more frequently found in semen samples from asymptomatic infertile patients than in those from fertile men. Bacteriospermia is also a common problem of male partners from couples undergoing IVF. Therefore, the effects of microorganisms on human sperm acrosome reaction of oocytes have been studied in vitro and in vivo. Incubation of spermatozoa with Escherichia coli or Mycoplasma hominis in vitro resulted in reduced sperm motility and inducibility of acrosome reaction (delta AR) after exposure to calcium ionophore A23187. To show possible effects of E. coli and mycoplasma species on sperm functions in vivo, data from 488 patients were evaluated, in whose ejaculates microbiological examinations and determinations of acrosome reaction after exposure to low temperature had been performed. U. urealyticum and E. coli were found in semen samples from 52 and 31 men, respectively. M. hominis was only present in a minor number of samples and was not included in this study. Semen concentrations of E. coli and U. urealyticum ranged between 500-100,000 cfu x ml-1 and 100-80,000 cfu x ml-1. No correlation was found between delta AR and concentration of bacteria (Spearman rank correlation coefficient, E. coli: r-0.081, P = 0.6644; U. urealyticum: r = -0.081, P = 0.5698). In 69% of cases with U. urealyticum infection and reduced inducibility of acrosome reaction, this sperm function was normal after antibiotic therapy. However, improvement of acrosomal function may only be due to intra-individual variations of acrosome reaction. While E. coli and mycoplasma species affect sperm functions in vitro, the present data and a review of the literature fail to demonstrate similar effects in vivo.

Regulation of the human leukaemia inhibitory factor (LIF) promoter in HEC-1B endometrial adenocarcinoma cells.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine which has been found to be expressed in the human endometrium and to play an important role in human reproduction. In the present study we investigated expression and regulation of the human LIF promoter in HEC-1B endometrial adenocarcinoma cells using a luciferase reporter plasmid bearing a 666 bp promoter fragment (h666LIF-Luc) in transient transfection assays. HEC-1B cells were first shown by reverse transcription-polymerase chain reaction (RT-PCR) to be able to produce endogenous LIF mRNA. The LIF promoter was efficiently transcribed in HEC-1B cells, showing much higher levels of basal activity than in the previously studied Jurkat T-lymphoma cells and SKUT-1B uterine mesodermal tumour cells. The activity of the LIF promoter was stimulated in HEC-1B cells by a combination of phorbol ester (TPA) and ionomycin, which we had previously found to strongly induce its activity in Jurkat T-lymphoma cells. We next studied the effect of progestin (medroxyprogesterone acetate; MPA) on the LIF promoter activity in HEC-1B cells. The LIF promoter was not stimulated by MPA treatment in the presence of transfected progesterone receptor B (PR-B) expression vector in HEC-1B cells, while we had previously described its induction by MPA in SKUT-1B cells. This indicates that progestin-dependent regulation of the LIF promoter in uterine tumour cells is different in cells of epithelial and mesodermal origin.

Expression of the apoptosis-inducing Fas ligand (FasL) in human first and third trimester placenta and choriocarcinoma cells.

The Fas (Apo-1/CD95) ligand (FasL) belongs to the tumor necrosis factor family and acts through its receptor (FasR/ Apo-1/CD95) to induce apoptosis in target cells. FasL is expressed in several immunologically privileged sites. Induction of apoptosis by FasL in invading lymphocytes acts as a mechanism of immune privilege and is important in preventing graft rejection. Furthermore, FasL is expressed in certain malignancies and it has been implicated as a possible key mechanism in immune privilege of these tumors. Since the invading placental trophoblast is another very important site with a privileged immune status, we investigated whether FasL is expressed in the normal and tumoral human placenta. For this purpose, mRNA was extracted from first and third trimester human placental samples as well as from JEG3 choriocarcinoma cells and reverse transcribed to obtain cDNAs. These were used as templates for PCR analysis of FasL expression, in which specific primers were employed to amplify an 853 bp fragment spanning the whole FasL coding region. A product of the appropriate length was amplified from normal placenta as well as from the choriocarcinoma cells. Expression of FasL protein was confirmed by Western Blot and was localized to trophoblast by immunohistochemistry using a FasL-specific antibody. Expression of FasL in the human placenta indicates that induction of apoptosis in lymphocytes by the invading trophoblast could be an important mechanism implicated in the immune tolerance of the fetal semi-allograft.

Progestin-dependent stimulation of the human leukemia inhibitory factor promoter in SKUT-1B uterine tumor cells.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which is essential for implantation in rodents and is expressed during the progesterone-dominated secretory phase of the menstrual cycle in the human endometrium. However, the effect of progestin on the transcriptional regulation of the LIF promoter has not been studied so far. In the present study, we used a luciferase reporter plasmid bearing 666 bp of the human LIF promoter (hLIF666-Luc) to investigate the effects of progestin on the transcriptional regulation of LIF in SKUT-1B uterine tumor cells. Jurkat T-lymphoma cells were used for comparison. Since both cell lines are devoid of functional progesterone receptors (PR), we co-transfected the cells with hLIF666-Luc and an expression vector for the human PR form B (PR-B) or A (PR-A). Addition of the progesterone agonist MPA (medroxy-progesterone acetate, 2.5 x 10(-7) M) resulted in induction of LIF transcription only in SKUT-1B cells, while it had no effect in Jurkat cells. Both PR forms were effective in inducing the LIF promoter in SKUT-1B cells when activated by MPA. However, the induction through PR-A was inhibited more efficiently by the progestin antagonist RU 486. We next investigated the stimulatory effect of MPA in SKUT-1B cells on deletion constructs (h274LIF-Luc, h148LIF-Luc and H82LIF-Luc) and found that it is maintained on these fragments. Thus, 82 bp are sufficient to mediate this effect. Our results show that the human LIF promoter is active in uterine tumor cells, and that it is differentially regulated by progestin in cells of uterine and lymphoid origin.

Transcriptional regulation of the human 'leukemia inhibitory factor' gene: modulation by glucocorticoids and estradiol.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine implicated in various pathological conditions, such as rheumatoid arthritis and osteoporosis. Despite its possible importance as a therapeutic target, very little is known about the regulation of human LIF. In particular, its regulation at the promoter level has not been studied so far, and was, therefore, the focus of the present work. After showing that Jurkat T lymphoma cells can be induced to express endogenous LIF mRNA, we used this cell line as a model to study the regulation of the human LIF promoter in transient transfection assays. For this purpose, a 666 bp fragment of the human LIF 5'-flanking region, amplified from genomic DNA by nested polymerase chain reaction (PCR), was used for the construction of a luciferase reporter plasmid (hLIF666-Luc). In unstimulated Jurkat cells, the human LIF promoter showed low constitutive activity. The promoter was induced upon stimulation with phorbol ester (TPA). Combined stimulation with TPA and the calcium ionophore ionomycin resulted in strong synergistic induction of luciferase activity from the LIF promoter. Transfection experiments with deletion constructs (hLIF274-Luc and hLIF82-Luc) located the region required for this induction to a 192 bp portion of the promoter, which carries two putative c-ets binding sites. We then investigated the effect of glucocorticoids and estradiol by cotransfecting the respective receptors. Both hormones strongly inhibited the stimulation of the LIF promoter by TPA and ionomycin. Since LIF is implicated in the pathogenesis of inflammatory and degenerative conditions, such as rheumatoid arthritis and osteoporosis, the finding that therapeutic agents employed in the treatment of such conditions, i.e. glucocorticoids and estrogens, can modulate the induction of LIF at the transcriptional level, is of particular interest.

Histamine log(dose)-response curves in asthmatics. A comparison between the Wiesbadener Doppelinhalator and the De Vilbiss Model 645 nebulizer.

In 9 asthmatic patients log(dose)-response curves were obtained on 4 successive days with the Wiesbadener Doppelinhalator (WDI) and the De Vilbiss (De V) 645 nebulizer, respectively. log(dose)-response was expressed as a quadratic regression equation. From those equations the dose, causing a fall in response (FEV1) of 10% of the initial value, was obtained and defined as the provocative concentration (PC(10] or sensitivity. Moreover, the reactivity was defined as the slope of the linear regression through the steeper part of the curve. Although we compared the De V and the WDI log(dose)-response curves after correction for the different liquid output, a significantly greater sensitivity was found for the De V nebulizer. As to the reactivity, no significant differences were found. The difference in sensitivity could perhaps be explained by the fact that, as compared with the De V nebulizer, the WDI may cause a larger deposition of aerosol on the throat and the pharynx, due to the much greater linear velocity of its aerosol jet.