Electric field-driven coherent spin reorientation of optically generated electron spin packets in InGaAs.

Full electric-field control of spin orientations is one of the key tasks in semiconductor spintronics. We demonstrate that electric-field pulses can be utilized for phase-coherent ±π spin rotation of optically generated electron spin packets in InGaAs epilayers detected by time-resolved Faraday rotation. Through spin-orbit interaction, the electric-field pulses act as local magnetic field pulses. By the temporal control of the local magnetic field pulses, we can turn on and off electron spin precession and thereby rotate the spin direction into arbitrary orientations in a two-dimensional plane. Furthermore, we demonstrate a spin-echo-type spin drift experiment and find an unexpected partial spin rephasing, which is evident by a doubling of the spin dephasing time.

Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium).

Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbiodinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endosymbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryopreservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers worldwide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stresses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotectants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D(2)O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similarities in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, suggesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appendix describes our H(2)O:D(2)O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability.

New injectable self-assembled hydrogels that promote angiogenesis through a bioactive degradation product.

Hydrogels used in regenerative medicine are often designed to allow cellular infiltration, degradation, and neovascularization. Low molecular weight hydrogels (LMWHs), formed by self-assembly via non-covalent interactions, are gaining significant interest because they are soft, easy to use and injectable. We propose LMWHs as suitable body implant materials that can stimulate tissue regeneration. We produced four new LMWHs with molecular entities containing nucleic acid and lipid building blocks and analyzed the foreign body response upon subcutaneous implantation into mice. Despite being infiltrated with macrophages, none of the hydrogels triggered detrimental inflammatory responses. Most macrophages present in the hydrogel-surrounding tissue acquired an immuno-modulatory rather than inflammatory phenotype. Concomitantly, no fibrotic capsule was formed after three weeks. Our glyconucleolipid LMWHs exhibited different degradation kinetics in vivo and in vitro. LMWHs with high angiogenic properties in vivo, were found to release glyconucleoside (glucose covalently linked to thymidine via a triazole moiety) as a common by-product of in vitro LMWH degradation. Chemically synthesized glyconucleoside exhibited angiogenic properties in vitro in scratch assays with monolayers of human endothelial cells and in vivo using the chick chorioallantoic membrane assay. Collectively, LMWHs hold promise as efficient scaffolds for various regenerative applications by displaying good biointegration without causing fibrosis, and by promoting angiogenesis through the release of a pro-angiogenic degradation product. STATEMENT OF SIGNIFICANCE: The main limitations of biomaterials developed in the field of tissue engineering remains their biocompatibility and vascularisation properties. In this context, we developed injectable Low Molecular Weight Hydrogels (LMWH) exhibiting thixotropic (reversible gelation) and thermal reversible properties. LMWH having injectability is of great advantage since it allows for their delivery without wounding the surrounding tissues. The resulting gels aim at forming scaffolds that the host cells colonize without major inflammation, and that won't be insulated by a strong fibrosis reaction. Importantly, their molecular degradation releases a product (a glycosyl-nucleoside conjugate) promoting angiogenesis. In this sense, these LMWH represent an important advance in the development of biomaterials promoting tissue regeneration.

Biophysics of zebrafish (Danio rerio) sperm.

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37+/-0.02 (SEM), an isosmotic cell volume (V(o)) of 12.1+/-0.2 microm(3) (SEM), a water permeability (L(p)) in 10% dimethyl sulfoxide of 0.021+/-0.001(SEM)microm/min/atm, and a cryoprotectant permeability (P(s)) of 0.10+/-0.01 (SEM)x10(-3)cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24h at 0 degrees C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.

Fatal anaphylactic sting reaction in a patient with mastocytosis.

We report on a 33-year-old female patient with indolent systemic mastocytosis and urticaria pigmentosa who died of an anaphylactic reaction after a yellow jacket sting. As she had no history of previous anaphylactic sting reaction, there was no testing performed in order to detect hymenoptera venom sensitization. But even if a sensitization had been diagnosed, no venom immunotherapy (VIT) would have been recommended. It is almost certain that VIT would have saved her life and it is most likely that VIT is indicated in some patients with mastocytosis with no history of anaphylactic sting reaction. However, no criteria have been established in order to allow a selection of mastocytosis patients eligible for such a 'prophylactic' VIT.

Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display.

Vascular beds are known to differ in structure and metabolic function, but less is known about their molecular diversity. We have studied organ-specific molecular differences of the endothelium in various tissues by using in vivo screening of peptide libraries expressed on the surface of a bacteriophage. We report here that targeting of a large number of tissues with this method yielded, in each case, phage that homed selectively to the targeted organ. Different peptide motifs were recovered from each of these tissues. The enrichment in homing to the target organs relative to an unselected phage was 3-35-fold. Peptide sequences that conferred selective phage homing to the vasculature of lung, skin, and pancreas were characterized in detail. Immunohistochemistry showed that the phage localized in the blood vessels of their target organ. When tested, the phage homing was blocked in the presence of the cognate peptide. By targeting several tissues and by showing that specific homing could be achieved in each case, we provide evidence that organ- and tissue-specific molecular heterogeneity of the vasculature is a general, perhaps even universal, phenomenon. Our results also show that these molecular differences can serve as molecular addresses.

Avian genetic resource banking: can fish embryos yield any clues for bird embryos?

Cryopreservation of avian germplasm is becoming better understood and more commonly practiced. However, one area that would be of great benefit for genome resource banking is the preservation of avian embryos. Little is know about the cryobiology of avian embryos, and they have never been successfully cryopreserved. However, it is likely that they share many of the challenges of other yolk-filled multicompartmental embryos. For example, the fish embryo has 1) a large overall size, resulting in a low surface-to-volume ratio, which retards water and cryoprotectant efflux/influx; 2) large-sized cells, such as the yolk, which could increase the likelihood of membrane disruption by intracellular ice formation; 3) compartments, such as the blastoderm and yolk, with differing permeability properties; and 4) susceptibility to chilling injury. Both the avian and fish systems share many physical and anatomical properties, and it is predicted that some of the same permeability barriers would exist in both as well. Although the systems are similar, some of the goals, and thus the practices, to protect the genome may be quite different. One of these major goals in avian developmental biology is to produce chicken:chicken transgenic animals, especially those with germ line transmission. Producing efficient germ line transmissions and being able to cryopreserve these transmissions would be extremely beneficial to both basic and agricultural science. This could be accomplished through the cryopreservation of embryonic gonadal tissue followed by grafting into a host. The gonadal/tail-graft system would provide an advantage for cryopreservation because it is small (in comparison with the whole embryo), has fairly uniform tissue, and contains the essential primordial germ line cells capable of recreating the genetic line of interest. Moreover, because the chicken is such a robust model for most other avian species, the cryopreservation of the gonadal/tail-graft may potentially open up similar treatments for other commercially important species.

The G2964A polymorphism of the STAT6 gene in inflammatory bowel disease.

BACKGROUND AND AIMS: Linkage of inflammatory bowel diseases to chromosome 12p13.2-q24.1 (IBD2) has been confirmed in several genome wide screens. The STAT6 gene is located within this chromosomal region. The transcription factor STAT6 is involved in the regulation of the TH1/TH2 immune response. Increased production of TH1 cytokines is crucial in the pathogenesis of Crohn's disease. PATIENTS AND METHODS: Therefore, we genotyped a single nucleotide polymorphism in the 3' untranslated region of the STAT6 gene (G2964A) in 243 patients with Crohn's disease, 100 patients with ulcerative colitis and 548 healthy controls. RESULTS: In comparison to controls, the G allele and the GG genotype frequencies were significantly increased only in Crohn's disease patients without a variation in the CARD15 gene (p<0.03 and p<0.02, respectively). CONCLUSIONS: Alterations in the STAT6 pathway may play a crucial role in the pathogenesis of distinct subgroups of patients with Crohn's disease.

Association between the promoter polymorphism T/C at position -159 of the CD14 gene and anti-inflammatory therapy in patients with inflammatory bowel disease.

Immune response to intestinal bacteria and genetic predisposition seem to play a crucial role in the pathogenesis of inflammatory bowel disease. A single nucleotide polymorphism in the promoter of the lipopolysaccharide-receptor CD14 gene (T/C at position -159) has recently been described. To evaluate the role of the CD14 gene in anti-inflammatory therapy, the functionally relevant T(-159)-->C promoter polymorphism has been genotyped in 72 patients with inflammatory bowel disease and associated with the cumulative steroid dose. Cumulative corticosteroid dose was significantly higher in ulcerative colitis patients with the TT genotype (2447.7 +/- 927.0 mg/yr) compared with the CT genotype (142.3 +/- 142.3 mg/yr, p=0.016) and the CC genotype (391.7 +/- 272.7 mg/yr, p=0.047). In contrast, in patients with Crohn's disease there was no significant difference of the cumulative corticosteroid doses between the various T(-159)-->C promoter CD14 genotypes. An altered immune response to lipopolysaccharides with influence on the anti-inflammatory therapy seems to play a role in the genetic predisposition to ulcerative colitis. Genetic stratification will lead to the development of individualized therapies in inflammatory bowel disease.

Expression of Ca2+-ATPase and Na+/Ca2+-exchanger is upregulated during epithelial Ca2+ transport in hypodermal cells of the isopod Porcellio scaber.

It is thought that a plasma membrane Ca(2+)-transport ATPase (PMCA) and a Na(+)/Ca(2+)-exchange (NCE) mechanism are involved in epithelial Ca(2+) transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca(2+)-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca(2+)-transporting condition to the stages of CaCO(3) deposit formation and degradation. During Ca(2+)-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.

Characterization of human papillomavirus 3 in warts of a renal allograft patient.

Multiple flat wart-like lesions of a renal allograft recipient were shown to contain HPV 3 or a serologically crossreacting virus by indirect immunofluorescence with monospecific animal antisera against HPV [1--5]. The patient's serum revealed virus specific antibodies (immunofluorescence titer 1/80). Papillomaviruses were isolated and after in vitro iodination 3 major proteins (MW 70.000, 56.000 and 43.000) were detected by SDS polyacryalmide gel electrophoresis. DNA was extracted from the warts and cleaved with the restriction endonuclease Hae III. Distinct bands were discernible within the background of cellular DNA and these fragments were identified as papillomavirus DNA by blot hypbridization with 32P-labeled viral DNA.

Monocytopoiesis in chronic eczematous diseases, psoriasis vulgaris, and mycosis fungoides.

Monocytopoiesis and blood monocytes were examined in 8 patients with disseminated chronic eczematous diseases, 8 patients with disseminated psoriasis vulgaris, and 8 patients with mycosis fungoides in plaque or tumor stage. Monocytopoiesis was moderately stimulated in all these patients. The stimulation manifested itself by: (1) a rise in relative number of promonocytes in bone marrow in all patients with eczema, in 1 out of 8 patients with psoriasis, and in 7 out of 9 examinations in patients with mycosis fungoides; (2) a rise in [3H]thymidine labeling indices of medullar promonocytes (8/8 eczema, 7/7 psoriasis, 8/9 mycosis fungoides); and (3) a rise in the naphthol-AS-D-chloroacetate esterase activity of blood monocytes, indicating premature monocyte marrow egress (3/5 eczema, 7/8 psoriasis, 9/9 mycosis fungoides). In eczema and psoriasis the mean enhancement of monocytopoietic activity was similar but less pronounced than in mycosis fungoides. In the latter disease there was no correlation between measured parameters and visible skin lesions. The results were interpreted as indicative of increased monocyte consumption by pathologic, immunologic, and/or inflammatory processes.

Correlation between human papillomavirus (HPV) type and histology of warts.

Forty warts from different patients and of different clinical type were examined histologically and virologically. Eight lesions were found to be associated with human papillomavirus type 1 (HPV 1), 15 tumors were induced by HPV 2, HPV 3 was detected 4 times, HPV 4 twice, and HPV 6 eleven times. HPV 3, HPV 4, and HPV 6 induced warts revealed a correlation between histology and virus type. They are characterized by the so called "edematous type clear cells". In HPV 3 associated flat warts pycnotic nuclei were mainly localized in the center of large vacuoles. In genital warts sickle shaped nuclei were pushed to the margin of the vacuolized cells. The histology of HPV 1 and HPV 2 induced warts was more heterogenous. With one exception HPV 1-induced lesions represented typical myrmecia warts, varying in the number and shape of inclusion bodies. HPV 2 associated common warts, however, revealed 3 very distinct histologic features: (1) Inclusion wart typical for HPV 1, (2) Classical common wart with marked condensation of keratohyalin granules, (3) Warts with extreme vacuolization of squamous and granular cells leading to a honeycomb-like picture.

Papillomavirus infection of the anogenital region: correlation between histology, clinical picture, and virus type. Proposal of a new nomenclature.

The clinical and histologic picture of 84 anogenital condylomatous and condyloma-like lesions of both sexes were analyzed in an effort to establish a correlation to the different papillomavirus (PV) types. The presence of human papillomavirus (HPV)-specific DNA sequences was confirmed through molecular hybridization and the presence of PV structure antigens was verified in thin sections by means of a group-specific anti-PV-antiserum using the peroxidase-antiperoxidase (PAP) technique. Three distinct clinical forms harboring distinct HPV types were distinguished: (1) Condylomata acuminata in which HPV-6 DNA was present in 37 of 59 samples and HPV-11 DNA in only 13 of 59 samples. HPV-16 DNA was not detected at all and 9 condylomatous lesions remained unclassified. (2) Flat condyloma-like lesions, where HPV-6 and HPV-11 were associated with lesions of low epidermal atypia in 8 and in 2 of 18 cases, respectively, and where HPV-16 was associated exclusively with 6 of 18 such lesions with severe atypia, called bowenoid papulosis. (3) Pigmented papules where HPV-16 was detected twice in lesions of bowenoid papulosis and HPV-11 in 2 of the benign pigmented lesions. The fourth clinical manifestation of genital papillomavirus infections--the so-called condylomata plana--was not available for virologic analysis. Histologically 5 different koilocytotic features were determined which could not be correlated either with one of the clinical pictures or with a specific PV type. HPV-16, however, was found frequently in non-koilocytotic lesions exhibiting the features of severe epithelial atypia known in bowenoid papulosis. The existence of PV structure antigens in these lesions could not be verified using the indirect immunoperoxidase--PAP-technique--in contrast to the koilocytotic lesions where clear evidence of the presence of HPV was proved in 36 of 56 (64.3%) of the cases.

Target molecules for anti-angiogenic therapy: from basic research to clinical trials.

There is growing evidence that anti-angiogenic drugs will improve future therapies of diseases like cancer, rheumatoid arthritis and ocular neovascularisation. However, it is still uncertain which kind of substance, out of the large number of angiogenesis inhibitors, will prove to be a suitable agent to treat these human diseases. There are currently more than 30 angiogenesis inhibitors in clinical trials and a multitude of promising new candidates are under investigation in vitro and in animal models. Important therapeutic strategies are: suppression of activity of the major angiogenic regulators like vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF); inhibition of function of alphav-integrins and matrix metalloproteinases (MMPs); the exploitation of endogenous anti-angiogenic molecules like angiostatin, endostatin or thrombospondin. Given the wide spectrum of diseases which could be treated by anti-angiogenic compounds, it is important for today's clinicians to understand their essential mode of action at a cellular and molecular level. Here we give an in-depth overview of the basic pathophysiological mechanisms underlying the different anti-angiogenic approaches used to date based on the most recent fundamental and clinical research data. The angiogenesis inhibitors in clinical trials are presented and promising future drug candidates are discussed.

Retinal growth and cell addition during embryogenesis in the teleost, Haplochromis burtoni.

Neurogenesis of the developing embryonic retina is described for the African cichlid fish, Haplochromis burtoni, from 4 days post fertilization until all cell phenotypes are generated (day 7). Cell addition and differentiation both begin at the same absolute location which later becomes the central retina. As observed in most other vertebrates, cones and ganglion cells differentiate first, followed by amacrine and bipolar cells. Rod photoreceptors, which are added late, differentiate last. Changes in retinal thickness, retinal stretching, cell size, and cell density were measured during development. From day 4 through 7, there is an increase in retinal thickness largely due to the expansion of the inner plexiform layer (IPL) and outer nuclear layer (ONL). The inner nuclear layer (INL) decreases in thickness and there is a transient decrease in the density of cells in the scleral portion of the INL. Cells increase in size in the ganglion cell layer (GCL) and the vitread INL, decrease in size in the sclerad INL, and remain the same in the ONL. Changes in the density of the cell layers were observed: the density of ONL cells increased, the density of GCL cells decreased, and INL cells increased then decreased. From day 4 to day 6, eye growth is entirely due to cell addition because no retinal stretching was observed in the ONL or the horizontal layer. During this same developmental period, the pattern and rate of neurogenesis were measured in the differentiated portion of the retina by means of 3H-thymidine labeling. A small number of cell divisions within the differentiated INL precede the onset of cell divisions in the ONL. The number of 3H-thymidine labeled cells within the INL increases at a low rate consistent with an asymmetric pattern of cell division characteristic of stem cells. In contrast, cell divisions in the ONL increase exponentially, consistent with a symmetric pattern of cell division characteristic of progenitor cells. Double-label experiments (3H-thymidine and a rod specific opsin antibody) show that some of the symmetrically dividing cells in the ONL express the rod specific opsin within 2 days, suggesting that these dividing cells are rod progenitors. Although we do not hae conclusive evidence, these developmental processes support the hypothesis that stem cells within the INL could be the source of rod precursors in the embryonic teleost retina.

Effects of arsenic cell metabolism and cell proliferation: cytogenetic and biochemical studies.

Chromosome analysis of lymphocytes from patients who had been exposed to arsenic showed frequent structural and numerical aberrations, even with an interval of decades since the last exposure. The in vitro addition of sodium arsenate induced the same chromosome changes--even to extreme of chromosome pulverizations--upon lymphocyte cultures from healthy subjects. Radioactive incorporation studies showed that arsenate was able to inhibit dose-dependently the incorporation of radioactively labeled nucleotide in RNA and DNA. Beyond that, arsenic blocked the cells in the S- and G2-phase. A general explanation for the inhibitory effect of inorganic arsenic on cell metabolism is the known strong affinity of arsenic to enzymes, especially to those containing sulfhydryl groups.

The embryogenesis of rod photoreceptors in the teleost fish retina, Haplochromis burtoni.

Development of the retina, like that of other tissues, occurs via an orderly sequence of cell division and differentiation, producing the functional retina. In teleost fish, however, cell division and differentiation in the retina continue throughout the life of the animal in two distinct ways. Stem cells in a circumferential germinal zone at the periphery of the retina give rise to all retinal cell types and progenitor cells located throughout the retina in the outer nuclear layer (ONL) produce new rod photoreceptors. These processes in adult retina recapitulate in space the embryonic events responsible for forming the retina. Analysis of these events in an African cichlid fish, Haplochromis burtoni, confirmed that cone photoreceptors differentiate first, followed by rod photoreceptors. Correspondingly, at the margin of the eye, cone photoreceptors differentiate nearer to the margin than do rods. Control of photoreceptor production is not understood. Here we present the time of appearance and distribution pattern of GABA and vimentin which are candidates for the control of retinal cell division and differentiation. Antibody staining reveals that both GABA and vimentin exhibit unique patterns of expression during embryonic retinal development. Vimentin immunoreactivity is evident throughout the retina in a spoke-like pattern between developmental Days 4 and 7, as both cone and rod photoreceptors are being formed. GABA is expressed in horizontal cells between Days 5 and 7, corresponding to the onset of rod differentiation in time and in position within the retina. Moreover, the wave of GABAergic staining in the horizontal cells parallels the wave of rod differentiation across the embryonic retina of H. burtoni. Thus, GABA may play a role in the development of rod photoreceptors.

Bowenoid papulosis. Presence of human papillomavirus (HPV) structural antigens and of HPV 16-related DNA sequences.

This study reviews 39 cases of anogenital bowenoid papulosis lesions in 22 individuals of both sexes that were analyzed clinically, histologically, immunocytochemically, and virologically. Macroscopically, three different types of lesions were demonstrated: erythematous macules; papules (lichenoid and/or pigmented papules); and leukoplakialike lesions. Microscopically, bowenoid papulosis fulfills the criteria of a squamous cell carcinoma in situ. Much like oral precancers, three distinct growth patterns (flat, endophytic, and exophytic) could be differentiated, which did not correlate with the clinical aspect of the lesions. In only two (5.12%) of the 39 cases of bowenoid papulosis could structural antigens of papillomaviruses be detected immunocytochemically (peroxidase-antiperoxidase technique). The DNA from 12 lesions that were analyzed for the presence of papillomavirus-specific sequences hybridized stringently in all cases with the human papillomavirus 16 specific DNA probe labeled with phosphorus 32.

Cytolytic T cell reactivity against melanoma-associated differentiation antigens in peripheral blood of melanoma patients and healthy individuals.

Antigenic peptides derived from several differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs). To examine their potential role in tumour-directed immune responses in vivo, we determined CTL reactivity against seven antigenic peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens in the peripheral blood of 10 HLA-A2+ healthy controls and 26 HLA-A2+ melanoma patients. The influenza matrix peptide (GILGFVFTL) presented by HLA-A2.1 was used as a control peptide. CTL reactivity was assessed in a mixed lymphocyte 'peptide' culture assay. Reactivity against Melan A/MART-1-derived peptide antigens was readily detectable in both melanoma patients and controls. Reactivity directed against tyrosinase-derived peptide antigens was also detected in both melanoma patients and healthy individuals, but less frequently. A measurable response against gp100/Pmel17-derived antigens was found in 1/10 controls and in 1/26 of the melanoma patients. Reactivity against the influenza matrix peptide was common in both melanoma patients and controls. Our findings show that precursor CTLs against melanocyte differentiation antigens can be detected in peripheral blood of melanoma patients and healthy individuals. The pattern of CTL reactivity directed against melanoma-associated antigens does not seem to be altered in melanoma patients. Despite antigen-specific CTL reactivity, tumour growth was not prevented in melanoma patients and autoimmune phenomena were not detected in healthy individuals. It remains to be determined whether precursor CTLs recognizing melanocyte differentiation antigens can be activated by immunization and lead to effective tumour rejection in vivo.