Paired indirect immunoenzyme staining with primary antibodies from the same species. Application of horseradish peroxidase and alkaline phosphatase as sequential labels

Summary Paired indirect immunoenzyme staining based on primary antisera from the same species was performed sequentially without intermediate antibody elution. The first antigen was labelled brown by an immunoperoxidase procedure (either the two-stage indirect method, the unlabelled antibody peroxidase-antiperoxidase method, or the avidin-biotin bridge method using diaminobenzidine (DAB) and hydrogen peroxide as the substrates. The second antigen was labelled blue by applying a two-stage indirect immuno-alkaline phosphatase procedure using naphthol AS phosphate and Fast Blue BB salt as the substrate. In this way, polyclonal mucosal immunocytes were revealed in distinctly contrasting colours when stained for and light chains. Glucagon and somatostatin (D) cells in human pancreatic islets, and gastrin and D cells in human gastric antral glands, were likewise clearly differentiated. Conversely, a mixed colour appeared in some immunocytes after staining for and chains. However, unbalanced colour mixing was sometimes difficult to interpret, and additional experiments demonstrated that unwanted interactions could take place between the two sequences of reagents if the density of the DAB deposits was insufficient. These pitfalls were incompatible with unequivocal double staining in the same cell. Nevertheless, paired staining could be conveniently applied with the described procedures when prior knowledge had established that the antigens in question were located in separate cells.     

Ultrastructure des cellules de Leydig et des cellules de Sertoli au cours du cycle testiculaire du Canard Pékin

Résumé L'ultrastructure des cellules de Leydig et des cellules de Sertoli du testicule du Canard Pékin a été étudiée au cours de la phase printanière du cycle sexuel, soit de janvier à juillet. Parallèlement on a effectué chez les mêemes animaux la recherche histochimique de la ⁵-3 -hydroxystéroïdedeshydrogénase (⁵-3 -HSDH) ainsi que le dosage, par chromatographie en phase gazeuse des stéroïdes androgènes dans le plasma veineux périphérique et dans le testicule.Les cellules de Leydig du Canard possèdent les organites cytoplasmiques spécifiques des cellules stéroïdogènes (reticulum lisse, mitochondries à crêtes tubulaires) ainsi que d'autres structures souvent rencontrées dans ce type cellulaire (microfilaments, vacuoles, granules denses). Les cellules de Sertoli contiennent un reticulum agranulaire moins développé que celui des cellules de Leydig et, très rarement, des mitochondries à crêtes tubulaires. Ces divers organites cytoplasmiques subissent un cycle saisonnier. La différenciation du reticulum lisse et des crêtes mitochondriales tubulaires commence en janvier et atteint son optimum en mars. Leur régression s'amorce en avril; d'abord accompagnée de structures dégénératives transitoires; elle conduit à la dispartion totale de ces organites en mait. Aucun indice de nécrose n'est observé dans ces cellules. Histochimiquement, une activité ⁵-3 -HSDH est présente dans les cellules de Leydig et, à un degré moindre, dans les tubes séminifères. Son intensité varie au cours du cycle.La confrontation de l'étude morphologique avec les résultats des dosages hormonaux montre qu'il existe une bonne corrélation entre le développement puis la régression du reticulum lisse et des crêtes tubulaires des mitochondries ainsi que des critères histochimiques de la ⁵-3 -HSDH d'une part et l'évolution de la testostérone plasmatique et testiculaire d'autre part. De plus on observe une augmentation du rapport testostérone/⁴-androstènedione testiculaire parallèlement au développement des organites cytoplasmiques. Ces organites semblent donc bien impliqués dans la synthèse et la sécrétion de la testostérone chez le Canard.

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Ultrastructure of Leydig and Sertoli cells in the testicular cycle of the Pekin duckBiochemical and cytoenzymological correlations

Summary Leydig and Sertoli cells of the testis of the Pekin duck were studied ultrastructurally during the spring phase of the sexual cycle, from January to July. Simultaneously, in the same animals, ⁵-3 -hydroxysteroiddehydrogenase (⁵-3 -HSDH) activity was ascertained histochemically and androgenic steroids of the plasma and testes were assayed by gas-liquid chromatography.The Leydig cells of the duck possess cytoplasmic organelles specific to steroidogenic cells (smooth reticulum, tubular mitochondria) as well as other structures often found in this cell type (microfilaments, vacuoles, denses bodies). The Sertoli cells contain an agranular reticulum that is less developed than that of the Leydig cells, and rarely show mitochondria with tubular cristae. These various cytoplasmic organelles undergo a seasonal cycle. The differentiation of the smooth reticulum and the mitochondrial tubular cristae begins in January and reaches a maximum in March. They begin to regress in April, at first with transitory degenerative structures, and then by total disappearance of these organelles by May. No indication of necrosis is observed in the cells. Histochemically ⁵-3 -HSDH activity is present in the Leydig cells, and to a slightly lesser degree in the seminiferous tubules. The intensity varies during the cycle.The comparison of the results of the morphological study with the hormone assays shows that a good correlation exists with the development and regression of the smooth endoplasmic reticulum and tubular cristae in the mitochondria, as well as the histochemical criteria of the ⁵-3 -HSDH on one hand, and the levels of plasma and testicular testosterone on the other hand. In addition there is an increase in the ratio of testicular testosterone to ⁴-androstenedione which parallels the development of the cytoplasmic organelles. These organelles thus seem to be implicated in the synthesis and secretion of testosterone in the duck.

Nous tenons à remercier très vivement Mme G. Collenot qui nous a initiées aux techniques d'histoenzymologie et nous a très gentiment permis de faire cette partie de nos recherches dans son laboratoire. Nous remerçions également M. Claude Pennarun, photographe, pour son excellente collaboration.     

A demonstration of the advantages of immunostaining in the quantification of amyloid plaque deposits

Summary Extracellular amyloid deposits are a feature of both Alzheimer type dementia and the normal aging process. Quantification of amyloid plaque deposits may well be useful in distinguishing between the senescent changes associated with normal aging and the pathological processes underlying dementia. To determine the most reliable and reproducible method for visualisation of the amyloid we have compared conventional silver staining techniques with -amyloid immunocytochemistry on a large sample of post-mortem brain tissue from both demented (n=15, age range 60–87) and non-demented (n=65, age range 14–99) patients. The degree of amyloid deposition was rated on a four point scale and ratings for the two techniques were significantly correlated (P<0.01). However, the immunocytochemical approach has a number of distinct advantages for quantification. The antibody to -amyloid is highly specific and does not stain neurofibrillary tangles or background features, it is considerably more sensitive than silver staining in highlighting diffuse amyloid deposits and, perhaps most importantly, it produces high contrast staining which allows easier image digitisation and subsequent computer image analysis.     

Tetramethyl Rhodamine Methyl Ester (TMRM) is Suitable for Cytofluorometric Measurements of Mitochondrial Membrane Potential in Cells Treated with Digitonin

A new method for cytofluorometric analysis of mitochondrial membrane potential has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells thus represents only the probe accumulated in mitochondria. Fibroblasts and cybrids were used as a model cell systems and optimal conditions for digitonin treatment and staining by TMRM were described. The TMRM signal collapsed by valinomycin, KCN and antimycin A and FCCP titration was used to gradually lower and characterise the stability of . The method is suitable for sensitive measurement of in different types of cultured cells.     

Expression of receptor protein tyrosine phosphatase agr; mRNA in human prostate cancer cell lines

Receptor protein tyrosine phosphatase (RPTP) is transmembrane protein phosphatases, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTP mRNA in several normal human tissues. We further investigated the regulation of expression of RPTP mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5-dihydrotestosterone (DHT) down-regulates RPTP mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTP mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTP as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTP mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTP is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.     

A putative major gene for rust resistance linked with a RFLP marker in sugarcane cultivar ‘R570’

Inheritance of resistance to rust was investigated in the self progeny of the sugarcane cultivar R570 also used to build a RFLP genetic map. Resistance was evaluated through both field and controlled greenhouse trials. A clear-cut 3 (resistant) 1 (susceptible) segregation indicative of a probable dominant resistant gene was observed. This is the first documented report of a monogenic inheritance for disease resistance in sugarcane. This gene was found linked at 10 cM with an RFLP marker revealed by probe CDSR29. Other minor factors involved in the resistance were also detected.     

Tubulin isoform usage in maize microtubules

Summary Antibodies specific to two of the maize -tubulin isoforms and to the three subfamilies of maize -tubulins were used in immunofluorescence microscopy to determine where and into which microtubule (MT) arrays these tubulin isoforms are incorporated in maize plants. All the tubulins examined appear to be incorporated into MTs in at least some cell types, with the possible exception of subfamily II -tubulins, which have been found only in the form of diffuse, nonfibrillar staining. Whereas the -tubulins of subfamily I appear to be used constitutively, others are used much more selectively in the plant, with 2-tubulin found in microtubules only during sexual reproduction. If a particular tubulin is used in the MTs of a given cell type, it appears to be incorporated into all the MT arrays found in the cell.Abbreviations DMSO dimethyl sulfoxide - MT microtubule - Pipes 1,4-piperazinediethanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Adoptions expérimentales de larves entre des fourmis de genres différents:Leptothorax nylanderi Förster etSolenopsis fugax Latreille

Résumé En l'absence de son propre couvain,Solenopsis fugax a élevé des larves deLeptothorax nylanderi, à la température de 22°C. Les ouvrières deSolenopsis détruisirent une partie de ces larves mais nourrirent celles qu'elles épargnèrent; ces dernières grossirent lentement pendant cinq à six mois, sans atteindre le stade prénymphe. Lorsque les ouvrières deS. fugax et les larves deL. nylanderi furent soumises ensemble à un hivernage préalable, elles donnèrent les mêmes résultats que sans hivernage. La présence d'une jeune reine deSolenopsis fut défavorable aux larves deLeptothorax.Inversement,L. nylanderi fut capable d'élever, à la température de 22°C, des larves deS. fugax et de les amener jusqu'au stade adulte. En présence de leurs propres larves, les ouvrières deL. nylanderi détruisirent tapidement toutes les larves deS. fugax introduites dans leur nid. D'autre part, un jeune couvain deLeptothorax remplaçait plus ou moins rapidement les larves deLeptothorax enlevées au préalable; sa présence était alors défavorable au développement des larves deSolenopsis. Un hivernage en début d'expérience fut plutôt favorable auxS. fugax, de même que la présence d'une reine féconde deLeptothorax. LesSolenopsis ainsi obtenus n'ont pas vécu plus de sept semaines. Ils étaient tous de caste ouvrière et de taille très petite.

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Summary When its own eggs and larvae missed,Solenopsis fugax bred larvae ofLeptothorax nylanderi, at a temperature of 22°C. TheSolenopsis workers killed some of this larvae and fed the others; these slowly grew bigger during five or six months but never reached the pre-pupa stage. The result was the same if the workers ofS. fugax and the larvae ofL. nylanderi overwintered together or not at all. A youngSolenopsis queen being there was noxious to the larvae ofLeptothorax.On the contrary,L. nylanderi has been able to breed larvae ofS. fugax up to the imago stage, at a temperature of 22°C. When its own larvae were in the nest, together with larvae ofS. fugax, the workers ofL. nylanderi killed the larvae ofS. fugax. On the other hand, new eggs and young larvae ofLeptothorax had to replace, more or less quickly, the larvae which had been taken away, and that was noxious to the growth ofSolenopsis larvae. An overwintering at the beginning of the experiment was rather favourable toS. fugax as was the presence of a fecundLeptothorax queen. TheSolenopsis thus obtained lived no longer than seven weeks. They all were workers and very small.

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S. Fugax L. Nylanderi 22° . Leptothorax , , , , . . S. Fugax Leptothorax.,L. Nylanderi 22° S. Fugax . L. Nylanderi ( )Leptothorax ; S. Fugax Solenopsis, Leptothorax. S. Fugax . .

     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

The assignment of a Thinopyrum distichum (Thunb.) Löve-derived translocation to the long arm of wheat chromosome 7D using endopeptidase polymorphisms

Summary Endopeptidase zymograms of the translocation line Indis revealed the presence of several major and minor bands that had differential expression in coleoptile and seed tissues. While Indis lacks Ep-D1a, which is present in the parental cultivar Inia 66, it also may not express any of the Th. distichum bands. The Indis zymogram was found to be identical to that of an isogenic line of Inia 66 possessing Lr19. Since the absence of an Ep-D1a product appears to be linked to the 7DL translocation, it is possible to use the null condition as a marker for both the Lr19 or Indis translocations. The Indis translocation also did not show recombination with the cn-D1 chlorophyl mutant on 7DL, confirming that a part of 7D was involved. The results of a telocentric mapping experiment involving the 7D telosomes indicated that in Indis a chromosome segment from Th. distichum replaced a large section of 7DL of Inia 66.     

Nucellar callus of ‘Femminello’ lemon,selected for tolerance toPhoma tracheiphila toxin,shows enhanced release of chitinase and glucanase into the culture medium

Phoma tracheiphila is the causative agent of the disease mal secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogenP. tracheiphila and its toxin. Some cultivars (e.g., Femminello lemon) are inherently sensitive while others (e.g., Tarocco orange) are tolerant. Cell lines derived from nucellar tissue of Femminello, Tarocco and a cell line selected for tolerance to the fungal toxin (Femminello-S) were used to study host-pathogen interaction. Our results showed that calli or conditioned media of Tarocco and Femminello-S inhibited the size of co-cultivated fungal colonies when compared to Femminello. In addition, conditioned medium of Tarocco as well as FemminelloS, but not Femminello, promoted bursting of hyphal tips. A ten-fold increase in chitinase and glucanase enzymatic activity, as evaluated by radiometric assay and laminarin hydrolysis respectively, was detected in Femminello-S extracellular extracts as compared to Femminello. An increase in chitinase was also shown by immunoblot analysis. Our findings suggest a positive correlation between the presence of chitinase and glucanase in the conditioned media of the cultured cells and the tolerance of those cells toP. tracheiphila toxin.     

Internalization of glial cell-derived neurotrophic factor receptor GFR alpha 1 in the absence of the ret tyrosine kinase coreceptor

1. Glial cell-derived neurothrophic factor (GDNF) interacts with a cell surface receptor, GFR1, that is linked via a glycosyl-phosphatidylinositol (GPI) lipid to the cell membrane. The neurotrophic activities of GDNF are mediated by binding to GFR1 and further interaction of the GDN–GFR1 complex with a coreceptor tyrosine kinase encoded by the c-Ret protooncogene. There is also evidence for the existence of cell signaling by GDNF and GFR1 in the absence of Ret.2. To further delineate the Ret-dependent and -independent functions of GDNF, cellular internalization of GDNF and GFR1 was examined in cells lines and primary neurons.3. Relative to other GPI-anchored receptors, efficient endocytosis ( 30–40% of total surface-bound ligand internalized after 2 min) of GNDF and GFR1 was observed in neuroblastoma and transfected-fibroblast cell lines that lack Ret. Primary hippocampal neurons from transgenic mice that express a wild-type GFR1 together with a mutant, tyrosine kinase-inactive Ret also internalized GDNF efficiently ( 20% of total surface-bound ligand internalized after 2 min). We also observed a ligand dependence for GFR1 internalization in the cell lines that lack Ret. Furthermore, a comparison in the presence and absence of Ret indicates that this coreceptor tyrosine kinase slows internalization at early time points.4. The data suggest different mechanisms of internalization for GDNF–GFR1 in the absence and presence of the Ret coreceptor.     

The ultrastructure of the accessory sex organs of the male rat

Summary The fine structure of the nuclei of epithelial cells of the dorsal lobe of the rat prostate were studied after administration of three different antiandrogenic compounds.The nucleolus appears to undergo a progressive disorganisation with partial fragmentation and dispersion of its normal components. Different types of intranuclear inclusions were found.The various alterations observed were often encountered within the same section. This may indicate that the nuclear alterations occur in the same compartment of the cell, and represent a dysfunction of integrated biochemical events occurring within this compartment.The findings support a view that the stimulatory secretory effect of androgens is mediated via a secretory center, located within the nucleolusassociated chromatin. Within this secretory center, the initial steps of the secretory process, the binding of the DHT receptor complex to DNA is assumed to occur.     

Sparse distribution of γ/δ T lymphocytes around human epithelial tumors predominantly infiltrated by primed/memory T cells

Summary Evidence from the mouse system has suggested that T lymphocytes accumulating in non-lymphoid tissue, in particular epithelia, may preferentially express the T cell receptor (TCR) . In this study, we characterize the T cell receptor or phenotype of lymphocytes infiltrating human tumors of epithelial origin using monoclonal antibodies (mAb) for immunohistology and flow cytometry on cells extracted by enzyme digestion. This report shows that the majority of CD3⁺ tumor-infiltrating lymphocytes are TCR ⁺ but a small percentage of TCR can be clearly defined scattered throughout the tumor tissue with apparently no microanatomical selection. So far there has been little evidence for an accumulation of activated T cells in human tumor tissues as defined by mAb against molecules appearing transiently during the acute phase of activation. Now mAb are available that can identify primed or memory T cells such as mAb UCHL-1 recognizing the CD45RO antigen. Here we show that CD3⁺ tumor-infiltrating lymphocytes have a statistically significant accumulation of primed T cells, as compared to the autologous peripheral blood lymphocytes, suggesting their immune stimulation by tumor cells.     

Electron microscopic examination of hepatic subcellular fractions for phosphatases

Synopsis Lead capture techniques have been investigated for demonstrating phosphatase activity, with the aid of the electron microscope, in subcellular fractions isolated from rat liver. Controls with no exposure to the enzyme substrate showed little staining (lead phosphate deposits). Results with preparations where the enzyme incubation period had been prolonged, and the lead phosphate deposits were heavy (over staining), were also reassuring: the staining did not tend to become generalized. Particularly satisfactory results were obtained when the tissue fractions were used in the form of unfixed suspensions for incubation with the substrate, this being performed with the minimum temperature and time needed to obtain representative staining of the elements present. Thus, for 5-nucleotidase in plasma-membrane fragments there is good staining after only 10 min at 2°C. The favoured concentration of lead ions is 2mm. When the reaction period has elapsed, glutaraldehyde is added, and the tissue material repelleted; only then should osmium tetroxide be added. Glucose-6-phosphatase staining, particularly in rough microsomes, showed a time-lag which could be abolished by prior sonication or treatment with deoxycholate. In general, the cytochemical findings tallied well with the biochemical characterization of the various fractions examined, including zonalrotor fractions containing smooth vesicles the origin of which could not be established merely by morphology.A. A. El-A. pursued the investigation (with pilot work at the Chester Beatty Research Institute and final experiments at the Courtauld Institute of Biochemistry, Middlesex Hospital, London W1) whilst on leave from the Cancer Institute, Faculty of Medicine, University of Cairo (present address).     

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms viability and culturability are often equated. However, in recent years the apparently self-contradictory expression viable-but-nonculturable (VBNC) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms viability and culturability in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces VBNC with expressions that are internally consistent.     

Ontogeny of estrogen receptor (ER) alpha and its co-localization with pituitary hormones in the pituitary gland of chick embryos

Estrogen is involved in regulating the development and hormone secretion of the anterior pituitary gland following its binding to estrogen receptors (ERs) expressed on pituitary cells. However, the pituitary is comprised of several cell types, and to date, there is no data about the specific cell types expressing ERs in embyonic chick pituitary. We therefore followed, by immunohistochemistry, the ontogeny of the pituitary ER alpha (ER), and the cell types expressing ER throughout chick embryo development. ER immunoreacitivity was restricted to the nuclei of pituitary cells. ER-immunopositive (ER⁺) cells were first detected at embryonic day 6.5 (E6.5), after which ER⁺ cells were consistently detected throughout the anterior pituitary gland, although the density of ER⁺ cells in the caudal lobe of the pars distalis was higher than that in the cephalic lobe. The proportion of ER⁺ cells in the pituitary was about 6% at E8.5; expression increased to 22% by E18.5 of gestation, with no additional change until hatching. Double-labeling of ER and pituitary hormones showed that the dominant cell types expressing ER were gonadotrophs immunopositive for luteinizing hormone (LH); the proportion of ER⁺ cells expressing LH increased throughout gestation and reached approximately 57% at hatching. About 2%–6% of thyroid-stimulating-hormone-immunopositive and 1%–2% prolactin-immunopositive cells expressed ER at later stages of embryonic development, but no growth-hormone-positive or adrenocorticotropic-hormone-positive cells expressed ER during the embryonic period. Thus, gonadotrophs are the main cell population expressing ER in the anterior pituitary gland of chick embryo, and ER is involved in regulating the development of the pituitary gland and the maturation of the hormone-secreting function.This work was supported by grants from the Natural Science Foundation for Outstanding Young Scientists of China (30325034) and the Natural Science Foundation of China (30170693, 30471264).     

Autologous tumor vaccines processed to express α-gal epitopes: a practical approach to immunotherapy in cancer

This review describes a method by which the human natural anti-Gal antibody can be exploited as an endogenous adjuvant for targeting autologous tumor vaccines to antigen-presenting cells (APCs). Tumor cells remaining in the patient after completion of surgery, radiation, and chemotherapy are the cause of tumor relapse. These residual tumor cells can not be detected by imaging, but their destruction may be feasible by active immunotherapy. Since specific tumor-associated antigens (TAAs) have not been identified for the majority of cancers, irradiated autologous tumor vaccines have been considered as an immunotherapy treatment that may elicit an immune response against the residual tumor cells expressing TAAs. However, tumor cells evolve in cancer patients in a stealthy way, i.e., they are not detected by APCs, even in the form of vaccine. Effective targeting of tumor vaccines for uptake by APCs is a prerequisite for eliciting an effective immune response which requires transport of the vaccine by APCs from the vaccination site to the draining lymph nodes. In the lymph nodes, the APCs transporting the vaccine process and present peptides, including the autologous TAA peptides for activation of the tumor-specific T cells. The required targeting of vaccines to APCs is feasible in humans by the use of anti-Gal. This antibody interacts specifically with the -gal epitope (Gal1-3Gal1-4GlcNAc-R) and is the only known natural IgG antibody to be present in large amounts in all humans who are not severely immunocompromised. The -gal epitope can be synthesized on any type of human tumor cell by the use of recombinant 1,3galactosyltransferase (1,3GT). Solid tumors obtained from surgery are homogenized and their membranes subjected to -gal epitope synthesis. Similarly, -gal epitopes can be synthesized on intact tumor cells from hematological malignancies. Administration of irradiated autologous tumor vaccines processed to express -gal epitopes results in in situ opsonization of the vaccinating cells or cell membranes due to anti-Gal binding to these epitopes. The bound antibody serves to target the autologous tumor vaccine to APCs because the Fc portion of the antibody interacts with Fc receptors on APCs. Since patients receive their own TAAs, the vaccine is customized for autologous TAAs in the individual patient. The repeated vaccination with such autologous tumor vaccines provides the immune system of each patient with an additional opportunity to be effectively activated by the autologous TAAs. In some of the immunized patients this activation may be potent enough to induce an immune-mediated eradication of the residual tumor cells expressing these TAAs.Abbreviations Ab Antibody - Ag Antigen - APC Antigen-presenting cell - DC Dendritic cell - FcR Fc receptor - -gal epitope Gal1-3Gal1-4GlcNAc-R - 1,3GT -1,3-Galactosyltransferase - KO mice Knockout mice for 1,3GT - OVA Ovalbumin - SA Sialic acid - TAA Tumor-associated antigen     

Genetic diversity in relation to heterosis and combining ability in spring wheat

Summary Genetic diversity among ten varieties of spring wheat used as parents in a diallel cross was assessed through multivariate analysis (D²-statistics) and then related to heterosis and SCA effects of their hybrids. The parents fell into three groups. Group I contained the varieties, Nobre, Girua and Carazinho; group II contained Sonalika, Lyallpur and Pitic 62 and group III contained Indus 66, Balaka, Sonora 64rs and MSl. The varieties of group I were good general combiners, while the varieties of group III were poor combiners. Significant heterotic and SCA effects for yield and yield components were observed in the hybrids of the parents belonging to different groups but not in the same group. Genetic divergence between the parents had a positive relationship with heterosis and SCA effects of the hybrids.