Identification of two homologous antigenic peptides derived from L1 HPV-16 and 18 proteins specific for the HLA-B*3901 allele

Summary.  In this work we present evidence that the homologous peptides IHSMNSTIL and IHSMNSSIL derived from L1 HPV-16 and 18 proteins respectively, and with high specificity for the allele HLA-B^*3901, according with an algorithm prediction program, induced T cell stimulation in patients with advanced cervical cancer positive for HPV-16 or 18 infection and for the HLA-B^*3901 allele. Interestingly, T lymphocytes derived from a patient with HPV-18 infection and stimulated with the peptide IHSMNSTIL were capable to kill a cervical cancer cell line named Rova, derived from the tumor of the same patient. In addition, the cytotoxic activity was strongly increased when this cell line was previously treated with hrIFN-γ. These results suggest that the CTL immune response to L1 HPV-16 and 18 protein derived epitopes is maintained in patients with advanced cervical cancer within specific alleles, and opens the possibility that homologous epitopes may be used in the generation of prophylactic vaccines for cervical tumors bearing different HPV-types. Received March 4, 2002; accepted May 20, 2002     

Absence of group II phospholipase A2, a Paneth cell marker, from the epididymis

Paneth cell-like metaplasia has been reported in the epithelium of the epididymis and prostatic adenocarcinomas. We studied the expression of group II phospholipase A2 (PLA2), a marker of Paneth cell differentiation, in six orchiectomy specimens with Paneth cell-like metaplasia. Both immunohistochemistry for group II PLA2 protein and in situ hybridization for the mRNA of group II PLA2 gave negative results in all six cases but positive reaction for lysozyme. The results show that the cells of the Paneth cell-like metaplasia are not true Paneth cells.     

Autologous primary muscle-derived cells transfer into the lower urinary tract

The goal of these experiments was to establish the basic methodology for future clinical applications of muscle-derived cells (MDC) tissue engineering and gene transfer for the treatment of urological dysfunction. Primary MDC isolated via preplating techniques from adult female SD rats were transduced with retrovirus encoding the expression of beta-galactosidase reporter gene. The MDC were injected into the right and left lateral walls of the bladder and proximal urethra of the autologous animals (n = 6) with a 10 microl Hamilton micro syringe. The amount of injected MDC ranged from 1 to 2 x 10(6) cells. The injected tissue was harvested after 7, 14, and 28 days, sectioned and examined histologically for beta-galactosidase and immunohistochemically for fast myosin heavy chain specific to skeletal muscle. The tissues were also stained for anti-CD4 and anti-CD8 antibodies to assess for cellular immune reaction. We have detected a large number of autologous MDC expressing beta-galactosidase and positively stained for fast myosin heavy chain in the bladder and urethral wall. Many injected myoblasts and myotubes were also seen in the bladder and urethral wall at each time point. Staining of lymphocytes with anti-CD4 and anti-CD8 antibodies was negative after MDC injection at each time point. We have demonstrated the long-term survival of autologous MDC and MDC mediated gene transfer into the bladder and urethral wall. Autologous MDC and MDC mediated gene transfer may be a promising treatment to augment bladder and urethral sphincter function.     

Glial fibrillary acidic protein (GFAP) immunochemical profile after Junin virus infection of rat cultured astrocytes

After five steps of purification including gel permeation, anti-angiotensin I affinity column chromatography followed by reverse-phase HPLC, a peptide immunoreactive to two different antisera (anti-angiotensin I) was purified to homogeneity from extracts of the leech Theromyzon tessulatum. The first 14 amino acid residues of the purified peptide (DRVYIHPFLLXWG) established by automated Edman degradation, reveal the existence in leeches of an angiotensin I-like molecule close to human angiotensin I. The sequence of the purified peptide presents 78.5% of homology with the N-terminal part of human angiotensin. Moreover, in its sequence, this peptide presents the cleavage sites of vertebrate angiotensin metabolic enzymes, i.e. the renin and the angiotensin-converting enzyme. This finding constitutes the first biochemical characterization of an angiotensin I in Invertebrates. It also reflects the high conservation of angiotensins in the course of evolution, suggesting a fundamental role of this family in fluid homeostasis.     

Intracellular lectin-binding sites in symbiont-bearingCrithidia species

Crithidia oncopelti, C. deanei, andC. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectinsUlex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, andCanavalia ensiformis, which recognize -l-fucose, - and -N-acetylglucosamine, -galactose and -N-acetylgalactosamine, -galactose, -galactose, -N-acetylgalactosamine, sialic acid and -d-mannose, and -d-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.     

Determination of capillary leakage due to recombinant interleukin-2 by means of noninvasive conductivity measurements

Summary One of the most common side effects of treatment with recombinant interleukin-2 (IL-2) is capillary leakage. Its genesis is not completely understood. The aim of the study was to determine whether capillary leakage can be monitored by means of a non-invasive conductivity technique and to study its starting point. Eight patients with advanced renal cell cancer were studied in a medium care section of the Department of Medical Oncology, University Hospital over 4 days during treatment sessions of continuous, intravenously administered IL-2 (mean dose of 15.6 × 10⁶ IU · m^–2 · day ^–1). The fluid shift from the intravascular to the extra- and intracellular compartments was monitored by means of noninvasive conductivity measurements. Changes in blood volume were calculated from serial erythrocyte counts. The clinical parameters of capillary leakage (oliguria, positive fluid balance, and gain in mass) were recorded. The mean gain in mass was 9% after 4 days of IL-2 treatment. The extracellular fluid volume increased significantly [46 (SD 23.2)%; P < 0.01], whereas the intracellular fluid volume did not change. The increase in blood volume (BV) amounted to 7% (P < 0.05). The decline in albumin concentration was significantly more than the increase in BV [38 (SD 4.3) %; P < 0.01], indicating capillary albumin leakage. The main changes were observed after the 2nd day of treatment. From this study, it is suggested that conductivity measurements are a suitable method to monitor capillary leakage induced by IL-2, and could be used to detect the exact onset and severity of this leakage. The leakage started within the first 24 h of treatment and was detected as a fluid shift from the intravascular to the extracellular space, while the intracellular compartment remained stable. These measurements could be useful during intervention studies with the aim of preventing this adverse effect of IL-2.     

Towards a novel classification of human malignancies based on gene expression patterns

As a result of progress on the human genome project, approximately 19 000 genes have been identified and tens of thousands more tentatively identified as partial fragments of genes termed expressed sequence tags (ESTs). Most of these genes are only partially characterized and the functions of the vast majority are as yet unknown. It is likely that many genes that might be useful for diagnosis and/or prognostication of human malignancies have yet to be recognized. The advent of cDNA microarray technology now allows the efficient measurement of expression for almost every gene in the human genome in a single overnight hybridization experiment. This genomic scale approach has begun to reveal novel molecular-based sub-classes of tumours in breast carcinoma, colon carcinoma, lymphoma, leukaemia, and melanoma. In several instances, gene microarray analysis has already identified genes that appear to be useful for predicting clinical behaviour. This review discusses some recent findings using gene microarray technology and describes how this and related technologies are likely to contribute to the emergence of novel molecular classifications of human malignancies.     

Estrogenic activity of estradiol and its metabolites in the ER-CALUX assay with human T47D breast cells

A number of metabolites of 17beta-estradiol were tested for their estrogenic activity using the ER-CA-LUX assay based on the increased expression of luciferase in exposed T47D breast cancer cells. E2beta and estrone showed similar potencies in the test, whereas E2alpha was 100 times less active. Incubation of cells with estrone (0.35 microM) resulted in the formation of E2beta, whereas the reverse reaction was observed for E2beta. The resulting equilibrium may explain the similar estrogenic potency of estrone in the test. The synthetic 17-hydroxy benzoate ester of E2beta was 3 times less active than the parent compound. The 17-hydroxy palmitate and oleate esters of E2beta, were respectively 25 and 200 times less active than the parent compound. The 2-hydroxy metabolites of E2beta and estrone showed a 5,000 to 10,000 fold lower activity. The 4-hydroxy metabolites were more potent than the 2-hydroxy metabolites, showing only a 20-200 times lower activity. The 2- and 4-methoxyesters of estrone were 700 times less active. It is concluded that the estrogenic potency of metabolites formed in cattle after treatment with E2beta, like estrone, E2alpha and especially the esters of E2beta, may be significant with respect to the potential risk of the use of estradiol for growth promotion in domestic animals in certain countries.     

Navigation within host tissues: cercariae orientate towards dark after penetration

The survival of skin penetrating cercariae depends on information on the direction to move toward deeper layers in the epidermis (the direction of further migration) and toward the surface (direction which must be avoided when migrating). We tested the hypothesis that parasites can use their photo-sensitivity for orientation away from the light-exposed skin surface towards darker locations. Cercariae of species invading humans (Schistosoma mansoni), birds (Trichobilharzia ocellata) and fish (Diplostomum spathaceum) oriented towards light sources when free swimming in cuvettes. However, they shifted to a negative photo-orientation when migrating in agar substrates after penetration and transformation to schistosomula. This is a first hint that parasites may use photo-orientation when they navigate in host tissues.