Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Possible bases of pseudoparasitism in Spodoptera littoralis larvae stung by Microplitis rufiventris

The effects of host age and parasitoid female age on the occurrence of 'Pseudoparasitism', using the Spodoptera littoralis-Microplitis rufiventris host-parasitoid system were investigated. The first four larval instars of the host are not equally suitable for parasitoid development. The proportion of pseudoparasitized hosts significantly increases when: (1) the age of the female parasitoid increases; (2) oviposition occurs mostly in fourth instar larvae; (3) a later age of the host instar is used; (4) the mandibles of the newly hatched parasitoid larvae mistakenly attack host interior organs (e.g. Malpighian tubules); and (5) an imperfect growth pattern of teratocytes occurs. The reluctance of female wasps to parasitize fourth instar host larvae is not due to the thickness of host cuticle but possibly due to the unfavourable physiological state of the host larvae. The age of host larvae at the time of parasitization may influence the adverse effects of parasitoid factors (e.g. polydnavirus, venom and teratocytes) on the growth of host larvae. It is suggested that females of M. rufiventris are able to determine the suitability of a potential host instar for the development of their offspring. The cell diameter of M. rufiventris teratocytes increases with increasing age of host larvae at the time of oviposition. The association within the host of living parasitoid larvae and functional teratocytes may be important for the survival of each other and consequently for successful parasitism.     

Dose-dependent influence of Microplitis rufiventris factors on developmental rate and growth of last-instar Spodoptera littoralis larvae

The solitary endoparasitoid, Microplitis rufiventris, attacks and can develop in earlier instars of Spodoptera littoralis larvae with preference to third‐instar larvae. We used the last stadium (sixth instar), a stage which is not naturally parasitized. The newly moulted larvae (0–3 h old) of this stadium were more acceptable for parasitization by the wasp females than the older ones (24 h old). Parasitization by M. rufiventris wasp of last instar S. littoralis larvae leads to dose (no. of eggs + parasitoid factors)‐dependent effects which were more pronounced at 20°C than at 27°C. A single oviposition into a sixth instar host larva resulted in normal development of the host. However, superparasitization increased the proportions of developmentally arrested hosts and number of live wasp larvae. Development of supernumerary individuals of the parasitoid in the host larva leads to dose‐related adverse effects on host growth and development. The present study may provide interesting opportunities for studying the physiological bases of host–parasitoid interactions and parasitoid intra‐specific competition in the biological system considered.     

Seasonality in the occurrence of two lepidopterous olive pests in Egypt

Abstract Prays oleae Bern, (OM) and Palpita unionalis Hüb., (JM) are two of the most important pests in olive groves in Egypt. A 3‐year monitoring study using sex pheromone traps in semi‐arid and arid olive groves was performed. In the semi‐arid grove, flight pattern of the OM was the same as in other Mediterranean countries, but in dates concordating plant phenology. The moth completes three generations annually: the first flight is in March to April, the second is in May to June and the third occurs in August to October. In the arid olive grove, an interesting flight pattern was observed. First flight was always very close or overlapped with the second one with no male catches during August to October. However, eggs were present most of the season, indicating unusually high female presence and oviposition activity of the OM during the absence of males in the traps. Generally, moth densities were significantly lower in low fruiting years than in higher ones and were also lower in the arid olive grove than in the semi‐arid one. In contrast, JM males were present all season, exhibiting six to seven and three to four overlapping flight peaks in arid and semi‐arid olive groves, respectively. Moth densities were significantly higher in the arid olive grove than those in the semi‐arid one. This study shows that trapping location and fruit bearing year are characteristics that strongly affect the grove‐specific information needed to estimate correctly adult emergence and thus the timing of control measures.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Dispersal and field progeny production of Trichogramma species released in an olive orchard in Egypt

Dispersal ability and field progeny production of augmentative released biological control agents depend on ecological adaptations of the particular species or strains used. Four species of the egg parasitoid genus Trichogramma were compared aiming to select suitable candidates for control of lepidopteran olive pests. Three of them (T. bourarachae Pintureau and Babault, T. cordubensis Vargas and Cabello, T. euproctidis Girault) had been previously collected from olive groves, whereas the commercially available strain used (T. evanescens Westwood) was originally isolated from sugarcane fields. During five consecutive field releases in an olive orchard near Cairo, dispersal and/or progeny production of these species was monitored using sentinel eggs placed at different heights in the release tree canopies as well as in neighboring trees (“distance effect”). The cardinal direction of dispersal was random for T. euproctidis and T. evanescens. Significant higher parasitism occurred on sentinel eggs placed on the middle part of tree canopy and highest parasitism was observed in trees where wasps had been released. Field progeny production was highest for T. bourarachae, followed by T. euproctidis and T. cordubensis. T. evanescens propagated less under field conditions. Inter-tree dispersal of all species except T. bourarachae was limited and, for biological control, releasing material should therefore be distributed on each olive tree, preferably also at different levels of the canopy.     

Studies on three species of Trichogramma. I. Foraging behaviour for food or hosts

The foraging strategies for both food or hosts of three trichogrammatid species: Trichogramma evanescens, Trichogramma cacoecia and Trichogramma dendrolimi were compared using artificial patches of Sitotroga cerealella eggs. In all experiments, inexperienced and newly emerged wasps of < 1 h old were singly tested. When the females were allowed to land upon the hosts, the decision-making process for initial acceptance or rejection was species-dependent. The initial search in T. evanescens was for food, whereas T. dendrolimi or T. cacoeciae start to oviposit immediately after their emergence. When honey-deprived or undeprived females were each exposed to single patches for 20 or 60 min, variations in mean number of probing females 'drilling and ovipositing' were also species-dependent. Acceptance of host eggs by honey-deprived females and subsequent egg deposition were higher in both T. cacoeciae and T. dendrolimi than in T. evanescens . For all species, the probing of honey-undeprived females was higher than that of deprived ones. When the exposure period was prolonged to 24 or 48 h and the number of patches per female increased to three, most of the honey-deprived or undeprived T. evanescens females attacked one patch, and only a few of them attacked two patches. Foraging activity of honey-deprived females of T. cacoeciae was restricted to single patches, whereas most undeprived ones attacked more than one patch. In contrast, honey-deprived or undeprived T. dendrolimi females attacked more than one patch. The experiments showed that T. dendrolimi females have more powerful ovipositing urges than looking for food and that the opposite was the case for T. evanescens , with T. cacoeciae being intermediate.     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.