Small Tissue Fragments Research Articles

Cryo-isolation: A Novel Method for Enzyme-Free Isolation of Pancreatic Islets Involving In Situ Cryopreservation of Islets and Selective Destruction of Acinar Tissue

Background A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ. Methods Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to −160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose- stimulated insullin release assessment. Results As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). Conclusions An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods.

First Report of Damping-Off Caused by Cylindrocarpon pauciseptatum on Pinus radiata in Spain.

In the fall of 2009, damping-off of Pinus radiata seedlings was observed in a pine nursery in Sant Feliu de Buixalleu, Girona Province, northeastern Spain. Plants exhibited needle blight, extensive root necrosis, and root death. Root sections of symptomatic plants were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small fragments of discolored tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were placed at 25°C in the dark for 10 to 14 days, and all fungal colonies were transferred to PDA. A Cylindrocarpon sp. was consistently isolated from necrotic root tissues. Single-conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer agar (SNA) (2) at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed abundant mycelium, which varied from white-to-grayish brown or golden brown. On SNA, all isolates produced two-septate, (35-) 39.4 (-40) × (7.5-) 7.7 (-8.75) μm, and three-septate, (32.5-) 40.9 (-52.5) × (7.5-) 7.7 (-8.75) μm, macroconidia. Microconidia, one-septate macroconidia, and chlamydospores were not observed. Identity of these isolates was determined by a multiplex PCR system using a set of three pair of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a 117-bp product that was characteristic of Cylindrocarpon pauciseptatum Schroers & Crous. Morphological characteristics also supported this identification (4). Internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolate 1052 and deposited in GenBank (Accession No. HQ441248). This sequence was identical (100%) with the sequence of C. pauciseptatum (GenBank Accession No. HM036590). Pathogenicity tests were conducted with inoculum produced on wheat kernels that were soaked in distilled water in flasks for 12 h. Each flask contained 200 ml of kernels that were subsequently autoclaved three times after excess water was drained. Two fungal disks from a 2-week-old culture of C. pauciseptatum (isolate 1052) grown on PDA were placed aseptically in each flask. Cultures in flasks were incubated at 25°C for 4 weeks and shaken once a week. A plastic pot (220 cm3) was filled with a mixture of sterilized peat moss and 10 g of inoculum. A 1-month-old seedling of P. radiata was planted in plastic pots and placed in a greenhouse at 25 to 30°C in a completely randomized design with six replications. Controls contained sterile wheat kernels. The experiment was repeated. Symptoms developed 20 days after inoculation and consisted of root lesions, a reduction in root biomass, needle blight, and the death of all seedlings. The fungus was reisolated from affected seedlings. Damping-off was not observed on the control plants. C. pauciseptatum causing black foot disease of grapevine (3) was first found in Spain in 2008, but to our knowledge, this is the first report of C. pauciseptatum causing damping-off of P. radiata in Spain.

Inductive activity of the posterior tip of planula in the marine hydroid Dynamena pumila

Activity of organizer regions is required for body plan formation in the developing organism. Transplanting a fragment of such a region to a host organism leads to the formation of a secondary body axis that consists of both the donor's and the host's tissues (Gerhart, 2001). The subject of this study, the White Sea hydroid cnidarian Dynamena pumila L. (Thecaphora, Sertulariidae), forms morphologically advanced colonies in the course of complex metamorphosis of the planula larva. To reveal an organizer region, a series of experiments has been performed in which small fragments of donor planula tissues were transplanted to embryos at the early and late gastrula stage, as well as to planulae. Only transplantations of a posterior tip fragment of a donor planula to a host planula of the same age led, in the course of metamorphosis, to the formation of a secondary shoot, which involved up to 50% of the host's tissues. After transplantations of tissue fragments of the anterior tip and the middle of the planula body, the formation of any ectopic structures was never observed. It was concluded that the posterior tip of the planula has organizer properties in Dynamena.

Preparation technique for cytologic study of EUS-guided FNA samples when onsite cytopathologist is absent: Cell block, smear, or both?

176 Background: Smearing EUS-guided fine-needle aspiration (FNA) samples, with or without cell block, is most commonly performed for cytologic study in centers without onsite cytology. The diagnostic yield of such technique is often low (35%-50%) and may be related to loss of diagnostic material during the smearing process. Cell block technique captures not only aspirated cells but also small tissue fragments, improving diagnostic yield and enabling additional ancillary studies. This study aimed to compare the diagnostic yield of cell block alone against conventional smear (±cell block) technique in the EUS-guided FNA evaluation of pancreatic solid mass without onsite cytological assessment. Methods: Data on the diagnostic yield of cell block alone technique performed over the last 12 months were prospectively collected and were compared to the yield of conventional smear ± cell block technique. A positive diagnostic yield was defined by the presence of adenocarcinoma and/or FNA findings consistent with the final surgical diagnosis. Results: Data were available for 96 patients, of which 66 had cell block alone and 30 had smear +/-cell block preparation. The diagnostic yield of cell block alone was significantly higher that that of smear +/- cell block (53/66 vs 13/30; P=0.003). The addition of cell block after smearing did not improve the diagnostic yield (smear: 42% vs. both: 45%, p=0.88). Of patients who had smear +/-cell block, the diagnostic yield was significantly higher when the smearing was performed by cytopathologist as compared to that by nurse (10/16 vs. 3/14, p=0.02). There was a direct relationship between the rate of diagnostic yield and the number of FNA passes performed. The yield was highest when 4 or more FNA passes was performed (85%) as compared to that of 3 passes (63%, p=0.049) and 2 or less passes (38%, p<0.0001). Conclusions: In the absence of onsite cytology services, the diagnostic yield from cell block alone was high (80%) and was superior to smear technique whether cell block was added to the evaluation of smears. In order to obtain adequate tissue material to maximise the diagnostic yield EUS-guided FNA of pancreatic mass, at least 4 FNA passes should be performed. No significant financial relationships to disclose.

Single nucleotide polymorphisms from cytochrome b gene as a useful protocol in forensic genetics against the illegal hunting of manatees: Trichechus manatus, Trichechus inunguis, Trichechus senegalensis, and Dugong dugon (Eutheria: Sirenia)

The identification of mitochondrial DNA polymorphisms is one of the most efficient methods for species differentiation. Genotyping of molecular markers using PCR/RFLP is a reliable, sensitive and inexpensive method for the detection of species specific mutations. The major causes of decline in Sirenia populations are accidental and intentional catches, collisions with boats and habitat loss. The goal of the present study was to identify, in silico, nucleotide mutations in the cytochrome b gene that can be used for the future development of forensic tools capable of using small tissue fragments to discriminate manatee meat from domesticated species meat commonly used as food sources (bovine, ovine, caprine and swine). DNA sequence alignments revealed two polymorphic sites distinguishing the manatee species from domestic species. The present study reinforced the power of cytochrome polymorphisms as powerful markers for species identification, which may be particularly useful for identifying vulnerable/endangered species. The data provided herein also suggest such mtDNA markers as important conservation tools for combating predatory manatee hunting for illegal meat trade in the Americas

Unusual case of uterine stump pyometra in a cat

This report describes an unusual case of uterine stump pyometra in a cat whose main clinical sign at presentation was abdominal straining. At the time of ovariohysterectomy, the surgeon reported that the uterine body had a purulent content. Nearly a month after the surgery the cat showed abdominal straining. The enlarged uterine stump, filled with purulent fluid, had caused a compression of the rectum and secondary intestinal sub-occlusion. Surgical revision consisted of draining the purulent content of the remnant of the uterine body and ablating as much of it as possible; checking of the ovarian pedicles revealed the presence of a small fragment of whitish tissue on the right side, which was shown to contain, by means of histological observation and immunohistochemical staining, ovarian tissue. Four months after surgical revision the queen did not show any pathological signs and 1 year later she is still in good health.

L’anatomie pathologique, une méthode d’étude originale des denrées alimentaires

To study the composition of foodstuffs (sausage, merguez, chipolata) on microscopic examination.Six sausages, merguez, and chipolatas, sold in supermarkets were studied. The samples were weighed before and after dehydration to assess the water composition. Foodstuffs specimens were formalin-fixed, paraffin-embedded and analyzed on microscopic examination. Proportions of different tissues were assessed by morphometric analysis.Specimens contained a high proportion of water (40 to 55%). Striated muscular fibers represented from 0.7 to 15.3% for the sausages and the merguez, and from 61 to 76.5% for the chipolatas. Sausages and merguez contained from 43.3 to 49.2% of adipose tissue. All the specimens had fibrous tissue and most of them had small fragments of bone and cartilaginous tissue. Fragments of salivar glands were found in the sausages and fragments of lymphoid tissue were found in merguez. There were neither parasite nor brain tissue. Manufacturer wrote on the label the presence of "meat" with no information about the nature and the proportion of tissues in the foodstuffs specimens. Prices of the foodstuffs were globally correlated to the quantity of muscular fibers in the specimens.Pathological studies are not performed in France for the control of foodstuffs. Microscopic analysis could be interesting, as well as biochemical and bacteriological studies, in order to identify the nature and the proportion of tissues involved in the composition of the foodstuffs, to search tissues with potential risk of pathogenic agents transmission, and to search for some parasites.

Histology of vitreous floaters, after removal by pars plana vitrectomy

Abstract Purpose To investigate the histopathology of vitreous floaters. Methods Ten consecutive (10) patients with vitreous floaters creating visual disturbance and affecting their daily life underwent 23G vitrectomy. First, a core vitrectomy was performed. If not already present, a posterior vitreous detachment was created. Next, a shaving of the vitreous base was done. All the aspirated fluid was collected in the vitrectomy cassette and fixed in Cytorich Red. After fixation, the fluid was centrifugated and the pellet was embedded in paraffin and sectioned and stained routinely for microscopy with H&E and PAS. Immunostainings for lymphocytes (CD3, CD20), macrophages (CD68) and retinal (nse) and glial tissue (gfap) were performed. Results In all cases sufficient material was collected for histology. The vitreous core consisted of fine fibrillar tissue in which floaters were recognised as a small fragment of glial tissue with cells with fibrillar cytoplasm, staining specifically for gfap. In most cases macrophages were randomly and diffuse present. Conclusion Vitreous floaters seem to originate from glial origin

The Number of Grafted Fragments Affects the Outcome of Testis Tissue Xenografting from Piglets into Recipient Mice

To optimize the procedure for testis tissue xenografting, we grafted 2, 4, 8, or 16 small fragments of immature porcine testis tissue under the back skin of immunodeficient castrated mice (n = 10 mice/group). At 8 months post grafting, the graft recovery rate did not differ between groups; however, not only the total but also the average graft weights were higher (by ∼12-fold and ∼2.5-fold, resp.) in mice receiving 16 fragments than those receiving 2 fragments (P < .05). The recipient mice with 16 fragments had the largest vesicular glands (indicators of testosterone release by the grafts) compared with those with 2 fragments (P = .007). The grafts in the group of 16 fragments also had more (P < .05) percentage of tubules with round spermatids than those of the group of mice receiving 2 fragments. Therefore, recipient mice can be grafted with at least 16 testis tissue fragments for optimal results.

Nerve Growth Factor Promotes the Survival of Goat Preantral Follicles Cultured in vitro

The aim of this study was to investigate the effects of nerve growth factor (NGF) on the in vitro culture of goat preantral follicles. Ovarian cortex fragments were cultured in α-MEM+ supplemented with 0, 1, 10, 50, 100 or 200 ng/ml NGF for 1 or 7 days. Small fragments of noncultured ovarian tissue as well as those cultured for 1 or 7 days were processed for histology and transmission electron microscopy. The results showed that after 1 or 7 days of culture at all concentrations of NGF, except at 1 ng/ml after 1 day of culture, there was a significant reduction in the percentage of normal follicles compared to noncultured tissues. At higher NGF concentrations (100 and 200 ng/ml) after 7 days of culture, there was a significant reduction in the percentage of normal follicles compared to tissues cultured in α-MEM+ alone or at the other concentrations of NGF. It is important to note that ultrastructural and fluorescent analyses confirmed only the integrity of follicles cultured with 1 ng/ml of NGF after 7 days. In contrast to noncultured control tissues, the percentage of developing follicles was significantly increased at all concentrations of NGF after 1 or 7 days of culture. We observed that follicular diameter was greater at 1 and 10 ng/ml NGF after culture for 7 days than at the other concentrations but was similar to follicles cultured in α-MEM+ alone. In conclusion, NGF improved the survival of goat preantral follicles cultured in vitro in a dose-dependent manner.

First Report of Fusarium semitectum as the Agent of Twig Cankers on Persian (English) Walnut in Italy.

English (Persian) walnut (Juglans regia) is the most widely cultivated walnut species. During the last 10 years, the increment of walnut cultivation in Italy has been accompanied by changes in cultural management. Changes were addressed to develop highly specialized cultivations with intensive pruning, fertilization, irrigation, and chemical treatments. For these reasons, more consideration has been given to the sanitary situation, in particular since 1998 when brown apical necrosis (BAN) was first observed (1). BAN is a fungal complex disease causing fruit drop, in which several Fusarium spp. are involved, among which Fusarium semitectum represents one of the major causal agents (2). From 2005 onward, investigations on sources of inoculum for BAN led to observing the presence of twig cankers on walnut trees cv. Lara located in northern Italy (Po valley). Cankers observed in late spring to summer were usually small (1 to 3 cm long) and mainly occurred on the new growth strongly incited by intensive pruning. Pale orange sporodochia were evident on lesions. Isolations were made from the margins of lesions, and small fragments of tissues (approximately 3 mm) were plated onto potato dextrose agar (PDA) after surface disinfection with 1% NaOCl. Whitish, light brown colonies were consistently obtained. On PDA, the production of fusoid, 1- to 3-celled mesoconidia was abundant. This characteristic was combined with the presence of two-spored polyphialides with a "rabbit-ear" appearance. Three to five septate macroconidia (38 × 4 μm) were produced in sporodochia on carnation leaf agar (CLA). On the basis of morphological characteristics (3), the fungus was identified as F. semitectum Berk. & Ravenel (synonyms F. incarnatum and F. pallidoroseum). Sequence comparison of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) was used to support the identification. A 99% identity for ITS was obtained with Accession No. AY633745 from Vietnam, while for TEF 1-α, a comparison was not available in GenBank. The sequences of one isolate (ISPaVe1946) were deposited in GenBank (Accession No. FN430680 for ITS and No. FN430737 for TEF 1-α). Pathogenicity tests were conducted outdoors on 1-year-old shoots of J. regia potted plants using ISPaVe1946 single-spored isolate. Mycelial plugs of 5-mm diameter, cut from the margin of PDA actively growing cultures, were placed under the bark and protected with Parafilm to prevent desiccation. Six inoculation points were performed. Controls were inoculated with plain PDA plugs. Within 2 months after inoculation, cankers developed in all inoculated points and were similar to those observed in nature. Controls showed no symptoms. Koch's postulates were fulfilled and the pathogen was constantly reisolated from lesions. To our knowledge, this is the first report of F. semitectum as the causal agent of twig cankers on walnut in Italy. This pathogen was already reported as an agent of canker on walnut in Argentina (4).

Abstract 4209: Organotypic multicellular glioma spheroids prepared from tissue fragments obtained by ultrasonic aspiration

Abstract Ultrasonic aspirators are commonly used to resect brain tumors because they allow safe, rapid and accurate removal of dissected tissue. However, the tissue fragments removed by ultrasonic aspirators are used surprisingly little in daily diagnostics and in clinical research. Due to the increasing need for vital human tumor tissue for these purposes, which today require multidisciplinary approaches, the aim of this study was to investigate whether tissue fragments obtained by ultrasonic aspiration could be cultured as organotypic multicellular spheroids. These spheroids have earlier been shown to represent an important alternative to commercially available cell lines having multiple mutations and phenotypic changes. Fragments obtained by ultrasonic aspiration (sonocut) from one anaplastic astrocytoma and seven glioblastomas and tumor biopsy tissue from four of these glioblastomas were included in the present study. Sonocut and tumor biopsy tissue were mechanically dissociated into small tissue fragments and cultured in a serum containing medium on agar-coated plates. If excess tissue was available the fragments were also cultured in a serum-free medium. After 12-18 days of culturing the tissue fragments had formed spheroids which were fixated and paraffin embedded. Hematoxylin eosin staining was performed in order to investigate the viability and morphology of the spheroids. Part of the material was stained immunohistochemically for the astrocytic marker GFAP and the blood vessel markers VWF, CD31 and CD34. In the present study all sonocut cultures contained viable spheroids, whereas one of the cultures obtained from tumor biopsy tissue did not. The spheroids were able to survive and proliferate under both serum and serum-free conditions. The spheroids from both sonocut and tumor biopsy tissue contained malignant traits including high cellularity, cellular pleomorphism, mitoses and necrosis. Some spheroids consisted solely of GFAP positive tumor cells, while others contained preserved blood vessels confirmed by VWF, CD31 and CD34 immunohistochemistry. In conclusion, we found that it was possible to obtain viable spheroids from sonocut-material under both serum and serum-free conditions. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4209.

First Report of Fusarium lateritium as the Agent of Nut Gray Necrosis on Hazelnut in Italy.

Hazelnut (Corylus avellana) is a traditionally cultivated nut species in Italy. Italy is the second largest producer of hazelnut in the world after Turkey. In early summer of 2000, a severe fruit drop (up to 60%) was observed in several hazelnut orchards located in the Latium Region in central Italy. The severity of yield losses led to investigating the etiology of the disease subsequently named nut gray necrosis (NGN) based on symptoms observed on the affected fruit. Symptoms consisted of a brown grayish necrotic spot/patch on the nut shell and bracts, sometimes involving the petiole (1). Isolations of the potential pathogen were from tissue that was sampled starting from bloom of female flowers to fully ripened fruit. Isolations from symptomatic tissue consisted of placing onto potato dextrose agar (PDA) small tissue fragments (approximately 3 mm) cut from the margin of lesions, while asymptomatic material (entire flowers or young fruit) was sectioned into small pieces. All the material was previously surface disinfected with 1% NaOCl. Slow-growing, dark grayish olive colonies were obtained consistently within 14 days of incubation at 20 to 22°C from symptomatic and asymptomatic material. Sporodochia were rarely produced on PDA, but never on carnation leaf agar. Dark grayish olive colonies were assigned to a Fusarium sp. Detached hazelnut fruit exposed to 20 μl of a mycelial suspension (105 CFU/ml) and incubated in a moist chamber at room temperature for 10 days produced orange sporodochia bearing 3 to 5 septate macroconidia (35 × 4 μm). On the basis of morphology, the fungus was identified as Fusarium lateritium Nees. (3,4). The identity was confirmed by internal transcribed spacer rDNA sequence comparison with BBA65248 (GenBank Accession No. AF310980). The sequences of two isolates, ISPaVe1874 and ISPaVe1976, were deposited in GenBank (Accession Nos. FN547420 and FN547445, respectively). Pathogenicity tests were performed in planta by inoculating, with the aforementioned isolates, young to fully formed fruit (approximately 24 mm in diameter) either with a drop of mycelial (106 CFU/ml) or conidial (106 conidia/ml) suspension. Drops were placed between the nut and leafy involucre. Controls were treated with sterile water only. Within 2 weeks after inoculation, a grayish necrosis developed on all the inoculated fruit and was similar to symptoms originally observed in the field. No differences were observed between the two methods of inoculation. On full-sized fruit, lesions extended from the shell to inner tissues. The pathogen was consistently reisolated from lesions. Controls showed no symptoms. To our knowledge, this is the first report of F. lateritium as the causal agent of nut gray necrosis on hazelnut in Italy, and this pathogen has never been reported as an agent of necrosis and drop of hazelnut fruit, but it was previously reported as an agent of twig cankers (2).

Epithelioid melanoma of the rectum: reappraisal of a deceptive microscopic simulator

We report 2 cases of rectal melanoma. The patients were a 48-year-old man and an 88-year-old woman. In both cases, the tumor presented as polypoid and necrotic masses, making initial diagnostic assessment difficult because of the regressive tissue changes. Microscopically, tumors were mostly composed of epithelioid, highly atypical cells mimicking poorly differentiated carcinoma or large cell lymphoma. Tumor cells were negative for keratin, leukocyte common antigen, and epithelial membrane antigen and positive for S100 protein and several melanoma markers, although in endoscopic biopsy specimens, the positive reaction was focal due to the limited viable tissue. One patient was treated with abdominoperineal resection. Pathologic examination of the surgical specimen revealed a polypoid mass infiltrating the muscle coat of the rectum. Local lymph nodes contained metastatic deposits. Microscopic examination of the rectal mucosa adjacent to the infiltrating neoplasm revealed colonization of the intestinal crypts by atypical melanocytes. This patient is alive 1 year after surgery with no evidence of recurrent disease. The other case was not deemed amenable to radical surgery because of the patient's advanced age and evidence of diffuse metastatic disease. She was alive 6 months after the initial diagnosis but was lost to follow-up. Data indicate that the diagnosis of melanoma, although feasible in large resection specimens, may be problematic in endoscopic biopsy, especially when only small tissue fragments are available and tumor regression hampers the accuracy of immunohistochemical stains. Documentation of intracryptic atypical melanocytes in nonneoplastic mucosa conceptually corroborates claims that rectal melanoma is related to preinvasive precursors as in cutaneous melanoma.

First Report of Leaf Blight and Root and Foot Rot of a Strelitzia Caused by Phytophthora nicotianae in Italy.

Bird of paradise, also known as crane flower (Strelitzia reginae Aiton), is a monocotyledonous flowering plant indigenous to South Africa. It is commonly grown and commercialized as an ornamental plant and it is appreciated for its beautiful flowers. In October of 2008, dark leaf spots and leaf blight associated with a severe root and foot rot were observed on several plants of S. reginae grown in a private garden located in Fiumicino, Italy. Small fragments of tissues (approximately 3 mm) collected from the base of leaves and roots and the margins of brown lesions, previously surface disinfected with 0.5% NaOCl, were plated onto potato dextrose agar (PDA) and incubated at 22°C in the dark. White, web-like, slow-growing colonies with coenocytic mycelium and hyphal swellings consistently developed from all plated tissue samples. Sporangia were ovoid or ellipsoid with prominent papillae (including some bipapillate) and frequently caducous with a short stalk. The dimensions of sporangia were 27 to 64 × 23 to 45 μm (average 43 × 35 μm). Chlamydospores were terminal or intercalary and approximately 30 μm in diameter. Isolates were considered heterothallic because they did not produce gametangia in vitro or in planta. On the basis of morphological features, isolates were identified as Phytophthora nicotianae (Breda de Haan). The identity was confirmed by internal transcribed spacer (ITS) sequence comparison in NCBI database with 99% identity with sequences available in GenBank (e.g., EU331089) and with cytochrome c oxidase subunit I (Cox I) with 99% identity with AY564196 by Kroon et al. (2). The sequences of one isolate, AB177, were deposited in GenBank (Accession Nos. FN430681 and FN552051 for ITS and Cox I, respectively). Pathogenicity tests were conducted in the greenhouse on leaves of a 1-year-old potted S. reginae plant by placing 5-mm-diameter mycelial plugs, cut from the margins of 10-day-old actively growing cultures, with mycelium in contact with plant tissues gently wounded with a needle. Controls were treated as described above, except that PDA sterile plugs were used. Plants were misted with water and placed in sealed plastic bags for 48 h. After 10 days, artificially wounded strelitzia leaves showed lesions (approximately 1 cm long). Controls remained symptomless. All inoculated leaves showed the same leaf symptoms as observed on naturally diseased plants. The pathogen was consistently reisolated from lesions. P. nicotianae has been reported as the causal agent of leaf blight and stem, collar, and root rot on several plants (1). It has been reported as an agent of Phytophthora blight on strelitzia in Japan (3). To our knowledge, this is the first report of P. nicotianae on strelitzia in Italy.

Effects of recipient mouse strain, sex and gonadal status on the outcome of testis tissue xenografting

The aim of the present study was to examine factors that may affect the outcome of testis tissue xenografting. Recipient factors were examined by grafting small fragments of testis tissue from newborn piglets under the back skin of immunodeficient mice of different strains (severe combined immunodeficiency (SCID) v. nude), sex (male v. female) and gonadal status (intact v. gonadectomised) using a factorial design (eight groups; n = 7 mice per group). Recipient mice were killed after 8 months to compare the gross and histological attributes of the recovered grafts. Overall, approximately 94% of grafts were recovered. Gonadectomy of male or female recipients did not affect any of the measured outcomes of testis tissue xenografting, therefore data were pooled. Overall, in terms of sex, male mice and, in terms of strain, SCID mice tended to show higher gross and histological development of grafts. The group of female nude mice had the lowest graft recovery rate (75%) compared with the other groups (95-100%; P < 0.05). The grafts from male SCID mice were, on average the largest and had the highest percentage of spermatozoa-containing seminiferous tubules among all the groups (P < 0.05). These results suggest that male SCID mice provide a suitable recipient model for testis tissue xenografting and that the mice do not need to be castrated for optimal results.

Tissue segregation of mitochondrial haplotypes in heteroplasmic Hawaiian bees: implications for DNA barcoding

The issue of mitochondrial heteroplasmy has been cited as a theoretical problem for DNA barcoding but is only beginning to be examined in natural systems. We sequenced multiple DNA extractions from 20 individuals of four Hawaiian Hylaeus bee species known to be heteroplasmic. All species showed strong differences at polymorphic sites between abdominal and muscle tissue in most individuals, and only two individuals had no obvious segregation. Two specimens produced completely clean sequences from abdominal DNA. The fact that these differences are clearly visible by direct sequencing indicates that substantial intra-individual mtDNA diversity may be overlooked when DNA is taken from small tissue fragments. At the same time, differences in haplotype distribution among individuals may result in incorrect recognition of cryptic species. Because DNA barcoding studies typically use only a small fragment of an organism, they are particularly vulnerable to sequencing bias where heteroplasmy and haplotype segregation are present. It is important to anticipate this possibility prior to undertaking large-scale barcoding projects to reduce the likelihood of haplotype segregation confounding the results.

Abstract ED01-04: Beyond IHC: Quantitative measurement of protein analytes on tissue slides

Abstract The large number and elusive nature of analytical and pre-analytic variables associated with immunohistochemistry (IHC) have relegated the technique to a semi-quantitative or qualitative status. However, as companion diagnostics become more critical to patient management, there is new motivation to move IHC to a fully quantitative assay. Toward this goal, we have developed the AQUA® method of quantitative immunofluorescence (QIF) to measure critical analytes in breast tissue. Using a series of cell lines which are standardized to recombinant protein quantified on western blot, we have measured estrogen receptor in three separate cohorts of breast cancer cases. The quantitative approach reveals a cutpoint of 50pg/ug total protein and suggests that conventional IHC may have a misclassification rate of around 15%. Furthermore, when comparing the misclassified cases, it appears that QIF classification predicts outcome more accurately than the conventional methods. As accurate as QIF is, it does not address preanalytical variables, most significantly including cold ischemic time, or the time between surgical removal of the tissue sample and fixation. A series of QIF methods for antibody validation may allow neutralization of this variable in assessment of tumor tissue. In summary, measurement of protein on a pathology slide can now be achieved with accuracy and reproducibility of nucleic acid assays or ELISA assays. Combining QIF with rigorous methods of standardization and antibody validation allows companion diagnostic tests to be done on very small tissue fragments. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):ED01-04.

First Report of Leaf Spot Caused by Pseudomonas cichorii on Coreopsis lanceolata in Italy.

Coreopsis lanceolata L. (Compositae), an ornamental species grown in parks and gardens, is very much appreciated for its long-lasting flowering period. In August of 2008, pot-grown plants with necrotic leaf lesions were observed in a commercial nursery located near Biella (northern Italy). Lesions were present, especially along the margin of basal leaves, and sometimes had a chlorotic halo. On infected leaves, dark brown necrosis developed. Leaf stalks were sometimes affected. In many cases, the leaves, especially those at collar level, were withered. Of 1,500 plants, 15% were infected by the disease. Microscopic examination did not reveal any fungal structures within the lesions. Small fragments of tissue from 30 affected leaves were macerated for 15 min in casein hydrolysate and 0.1-ml aliquots of the resulting suspension were spread onto Luria Bertani agar (LB) and potato dextrose agar (PDA). Plates were maintained at 22 ± 1°C for 48 h. No fungi were isolated from the leaf spots on LB or PDA. Colonies similar to those of Pseudomonas spp. were consistently isolated on LB. Colonies were fluorescent on King's medium B, levan negative, oxidase positive, potato soft rot negative, arginine dihydrolase negative, and tobacco hypersensitivity positive (LOPAT test). The bacterial colonies were identified as Pseudomonas cichorii (2). The internal transcribed spacer (ITS) region of rDNA was amplified using primers 27F and 1492R and sequenced (GenBank Accession No. FJ534557). BLAST analysis (1) of the 998-bp segment showed a 98% homology with the sequence of P. cichorii. The pathogenicity of one isolate was tested twice by growing the bacterium in nutrient broth shake cultures for 48 h at 20 ± 1°C. The suspension was centrifuged, the cell pellet resuspended in sterile water to a concentration of 107 CFU/ml, and 30 4-month-old healthy coreopsis plants were sprayed with the inoculum. The same number of plants was sprayed with sterile nutrient broth as a control. After inoculation, plants were covered with plastic bags for 48 h and placed in a growth chamber at 20 ± 1°C. Five days after inoculation, lesions similar to those seen in the field were observed on all plants inoculated with the bacterium, but not on the controls. Ten days later, 40% of the leaves were withered. Isolations were made from the lesion margins on LB and the resulting bacterial colonies were again identified as P. cichorii. The pathogen caused the same symptoms also on plants of Dendranthema frutescens (cv. Camilla), Chrysanthemum morifolium (cvs. Eleonora and Captiva), and an Osteospermum sp. (cv. Wild side) when artificially inoculated with the pathogen with the same methodology. The same bacterial leaf spot caused by P. cichorii was observed in 2005 in other nurseries in the same area on Phlox paniculata (3). To our knowledge, this is the first report of bacterial leaf spot caused by P. cichorii on C. lanceolata in Italy.

Application of an innovative technique for histological and histochemical study of different vegetal tissues

A new original "cryostabilization" technique (Dore, 1992; Dore et al, 2004) was applied to different animal tissues and organs. In this study, we apply this technique also to vegetal tissues (ripening and ripe fleshy fruits, buds and embryos). The proposed method preserves good tissue morphology and intracellular substances, and avoids loss of epicuticular waxes; moreover it maintains in situ some enzymatic activities. Being some samples very difficult to handle the original protocol had to be modified. The correct orientation in embedding of samples is an important requirement which is not easy to achieve when deadling with small fragments of tissues.