First report of root-knot nematodes (Meloidogyne species) infecting Chinese Elm (Ulmus parvifolia) in Florida, USA.

Samples of galled roots, resembling those induced by root-knot nematodes, and rhizosphere soil were collected from potted plants of Ulmus parvifolia cvs. Allee and Drake in Lake County, Florida. Nematode species were identified using both molecular analysis and morphology of perineal patterns. Meloidogyne enterolobii and M. javanica were identified from U. parvifolia cv. Allee. Meloidogyne arenaria and M. javanica were identified from U. parvifolia cv. Drake. This is a first report of these nematode species infecting Chinese Elm in Florida.Samples of galled roots, resembling those induced by root-knot nematodes, and rhizosphere soil were collected from potted plants of Ulmus parvifolia cvs. Allee and Drake in Lake County, Florida. Nematode species were identified using both molecular analysis and morphology of perineal patterns. Meloidogyne enterolobii and M. javanica were identified from U. parvifolia cv. Allee. Meloidogyne arenaria and M. javanica were identified from U. parvifolia cv. Drake. This is a first report of these nematode species infecting Chinese Elm in Florida.

First report of Meloidogyne enterolobii infecting Japanese blue berry tree (Elaeocarpus decipiens) in Florida, USA.

In October 2019, samples of galled roots with rhizosphere soil were collected from declining Elaeocarpus decipiens in Hernando County, Florida. Extracted root-knot nematodes were identified by both molecular and morphological methods as Meloidogyne enterolobii. This is a first report of this regulated root-knot nematode on Elaeocarpus decipiens in Florida.In October 2019, samples of galled roots with rhizosphere soil were collected from declining Elaeocarpus decipiens in Hernando County, Florida. Extracted root-knot nematodes were identified by both molecular and morphological methods as Meloidogyne enterolobii. This is a first report of this regulated root-knot nematode on Elaeocarpus decipiens in Florida.

Role of endoplasmic reticulum stress induction by the plant toxin, persin, in overcoming resistance to the apoptotic effects of tamoxifen in human breast cancer cells.

BACKGROUND: Persin is a plant toxin that displays synergistic cytotoxicity with tamoxifen in human breast cancer cell lines. Here, we examined the ability of persin to circumvent tamoxifen resistance and delineated the intracellular signalling pathways involved. METHODS: The induction of apoptosis in tamoxifen-resistant and -sensitive breast cancer cells was measured by flow cytometry following treatment with persin±tamoxifen. Markers of endoplasmic reticulum stress (ERS) were analysed following treatment, and their causal role in mediating persin-induced apoptosis was determined using chemical inhibitors and RNA interference. RESULTS: Cells that were resistant to an apoptotic concentration of tamoxifen maintained an apoptotic response to persin. Persin-induced apoptosis was associated with an increase in markers of ERS, that is, CHOP expression and XBP-1 splicing and was decreased by CHOP siRNA. The CASP-4 inhibitor Z-YVAD-FMK markedly inhibited persin-induced apoptosis in both tamoxifen-sensitive and -resistant cells. CONCLUSION: The cytotoxic effects of persin are CASP-4 dependent and mediated by CHOP-dependent and -independent ERS signalling cascades. Increased ERS signalling contributes to persin-induced reversal of tamoxifen resistance.

Prediction of HIV peptide epitopes by a novel algorithm.

Identification of promiscuous or multideterminant T cell epitopes is essential for HIV vaccine development, however, current methods for T cell epitope identification are both cost intensive and labor intensive. We have developed a computer-driven algorithm, named EpiMer, which searches protein amino acid sequences for putative MHC class I- and/or class II-restricted T cell epitopes. This algorithm identifies peptides that contain multiple MHC-binding motifs from protein sequences. To evaluate the predictive power of EpiMer, the amino acid sequences of the HIV-1 proteins nef, gp160, gag p55, and tat were searched for regions of MHC-binding motif clustering. We assessed the algorithm's predictive power by comparing the EpiMer-predicted peptide epitopes to T cell epitopes that have been published in the literature. The EpiMer method of T cell epitope identification was compared to the standard method of synthesizing short, overlapping peptides and testing them for immunogenicity (overlapping peptide method), and to an alternate algorithm that has been used to identify putative T cell epitopes from primary structure (AMPHI). For the four HIV-1 proteins analyzed, the in vitro testing of EpiMer peptides for immunogenicity would have required the synthesis of fewer total peptides than either AMPHI or the overlapping peptide method. The EpiMer algorithm proved to be more efficient and more sensitive per amino acid than both the overlapping peptide method and AMPHI. The EpiMer predictions for these four HIV proteins are described. Since EpiMer-predicted peptides have the potential to bind to multiple MHC alleles, they are strong candidates for inclusion in a synthetic HIV vaccine.

Identification of subdominant cytotoxic T lymphocyte epitopes encoded by autologous HIV type 1 sequences, using dendritic cell stimulation and computer-driven algorithm.

Conventional analysis of the cytotoxic T lymphocyte (CTL) response to HIV-1 may underestimate the true breadth of CTL epitopes recognized. This underestimation could be due to several reasons, including (1) the use of laboratory-adapted stains of HIV or consensus sequences, which would lead to the identification of only highly conserved epitopes, (2) the use of EBV-transformed B cells (B-LCLs) and vaccinia virus constructs in standard assays that may obscure low level CTL responses due to high EBV or vaccinia reactivity, and (3) relatively insensitive assays wherein PBMCs instead of professional APCs are used to stimulate CTL responses. To address these problems, we first identified an immunodominant HLA-B7-restricted CTL epitope, by standard cloning methods, in a long-term nonprogressor (LTNP). To determine whether the patient had CTLs specific for autologous viral sequences other than the dominant epitope, proviral DNA was cloned and sequenced. A matrix-based epitope algorithm (EpiMatrix) was used to identify the top 2% of peptides from the viral sequences with the highest likelihood of binding to HLA-B7. These 55 peptides were synthesized and tested for HLA-B7 binding in a T2/B7 cell line; 10 peptides were able to stabilize HLA-B7 on the cell surface. By using peptide-pulsed autologous dendritic cells as a more sensitive method of CTL stimulation, we found three additional subdominant CTL epitopes.

Cytogenetic study of solid ovarian tumors.

With the use of a short-term tissue culture method, 27 solid ovarian tumor specimens (from 22 patients) were successfully karyotyped. The majority of the specimens were from serous carcinoma (18 specimens, 2 of which were not invasive). Adenocarcinomas (two specimens), two endometrioid carcinomas, and one each of clear cell, mucinous, sarcoma, squamous carcinoma, and an unclassified sex cord carcinoma were also analyzed. The specimens showed marked cytogenetic heterogeneity, ranging from a normal karyotype (46,XX) to very grossly aneuploid, with multiple rearrangements. All chromosomes, excepting 13, 15, 19, 20, and 21 were positively identified in at least one rearrangement. Chromosomes 1, 6, and 7 were most commonly involved. Identified rearrangements were not limited to one carcinoma type. The most common deletions of 1p and 6q were identified in both serous carcinoma and adenocarcinoma. Deletion of 7q,(del(7)(q32)), was observed only in serous carcinoma but was limited to three patients. Correlations of modal chromosome count and number of marker chromosomes appeared to be associated with good prognosis for patients with serous carcinoma.

Analysis of inferred cytogenetic clonal evolution in a metastatic human ovarian carcinoma.

Cytogenetic analyses of solid tumors are hampered both by the difficulties in preparing high-quality banded metaphases and the high degree of karyotypic heterogeneity. Three specimens were obtained from a single patient with serous carcinoma for karyotyping and compared to determine whether the tumor was multifocal or metastatic. All three specimens were hypodiploid, sharing marker chromosomes, thus indicating metastasis. To simplify comparison between the specimens, a diagram of the inferred cytogenetic evolution of the cells analysed was developed. This method afforded a simple means of comparing the different tumor sites in a single patient and might also be applied to sequential samples from patients.

High quality metaphases from solid ovarian tumors.

Cytogenetic studies of human solid tumor tissue are hampered by poor quality preparations. Using a method of short-term tissue culture developed for ovarian carcinoma specimens, we have obtained large numbers of high quality metaphases suitable for analysis from 19 of 28 ovarian tumors studied.

Metabolic and developmental responses of preimplantation embryos to platelet activating factor (PAF).

Platelet activating factor (PAF) is an ether phospholipid produced by preimplantation embryos of a number of species. Production of PAF by embryos has been measured by detecting thrombocytopenia in a splenectomized mouse bioassay, platelet aggregation bioassays in vitro and a specific radioimmunoassay. Production is highly variable and is adversely affected by culture in vitro. It has, however, been correlated with morphology, development rates in vitro and the pregnancy potential of embryos following transfer. Investigations using PAF-antagonists have established an essential role for PAF in early pregnancy. Together with studies that have shown PAF to have direct effects on embryonic metabolism during culture in vitro, these observations suggest that PAF acts as an embryonic autocoid. Hence, a major site of action for embryo-derived PAF in vivo is the embryo itself. Supplementation of embryo culture media with PAF had no effect on the rate of development in vitro of 2-cell mouse embryos through to the blastocyst stage. However, PAF increased cell numbers of blastocysts cultured from the 2-cell stage and the mitotic index of embryos at both the 8-cell and blastocyst stages. Supplementation of culture media with PAF has also been shown to increase the implantation potential of both mouse and human embryos cultured in vitro. In the mouse, the effect of PAF in enhancing implantation rates was most evident when the developmental potential of untreated embryos was suboptimal. These observations suggest that the production of embryo-derived PAF is one limiting factor in maintaining the viability of embryos cultured in vitro.

Sterilization of a small caliber vascular graft with a polyexpoxy compound.

Sterilization of tissue based medical devices via cold sterilization processes has been limited to formaldehyde, glutaraldehyde, and mixtures of the same with alcohols and surfactants. The authors report the sterilization of a small caliber vascular graft with a combination of diglycidyl ether and ethanol. The sterilant contains 1-4% diglycidyl ether and 10-20% ethanol as an aqueous solution. Sterilization is achieved after exposure of the graft to the sterilant solution for a period of 7 days at an elevated temperature (30 degrees - 40 degrees C). The biologic indicator selected for efficacy studies was Bacillus subtilis niger ATCC 9372 (endospores). The grafts were inoculated with a concentrated endospore suspension and immersed in the sterilant solution for increasing time periods. After extensive rinsing over membrane filters to remove any residual sterilant, the grafts and filters were cultured in tryptic soy broth. D10 values were calculated using a fraction-negative, most probable number technique. Additionally, many representative bacteria and fungi were tested and found to be susceptible to the new sterilant developed. The diglycidyl ether/alcohol sterilant developed was found to be efficacious for sterilization of the tissue based vascular grafts tested.

Identification of PUMA as an estrogen target gene that mediates the apoptotic response to tamoxifen in human breast cancer cells and predicts patient outcome and tamoxifen responsiveness in breast cancer.

Recognition of the pivotal role of estrogen in the aetiology of breast cancer has led to the development of antiestrogens (AE), such as tamoxifen (TAM) as effective therapies for the treatment and prevention of this disease. However, despite their widespread clinical efficacy, response to AEs is often short-lived, and acquired or innate therapeutic resistance remains a major obstacle in the successful treatment of breast cancer. Thus, delineating the intracellular pathways that mediate the cellular response to estrogen could potentially lead to new, more effective approaches to the treatment of breast cancer, particularly endocrine-resistant disease. Here, we have identified the BCL-2 homology 3 (BH3)-only, pro-apoptotic regulator, PUMA (p53 upregulated modulator of apoptosis) as an estrogen target gene that is acutely downregulated in response to estrogen in breast cancer cell lines, independently of their p53 status. PUMA is transcriptionally upregulated following treatment with TAM, and knock down of PUMA expression in these cells attenuates the apoptotic response to TAM. Furthermore, low PUMA expression in breast carcinomas is significantly associated with breast cancer-specific death (P=0.0014 and P=0.0115, for mRNA and protein, respectively), and worse outcome in TAM-treated patients (mRNA, P=1.49e-05). These findings suggest that the dysregulation of apoptotic signaling pathways such as those executed through PUMA, can significantly impact on both the progression and therapeutic responsiveness of breast cancer. Moreover, they provide a convincing rationale for exploring new therapeutic approaches involving endocrine and non-endocrine therapies that target apoptotic pathways as an effective strategy for tackling endocrine refractory disease.

A case-control study of the pathology of oesophageal disease in systemic sclerosis (scleroderma).

BACKGROUND: Atrophy of the smooth muscle layers of the muscularis propria characterises oesophageal involvement in systemic sclerosis (scleroderma). The aetiology of this atrophy and of the resultant oesophageal dysfunction is unknown. OBJECTIVES: To examine oesophageal tissue for evidence of fibrosis, vascular disease, inflammatory reactions and neural abnormalities to determine the possible causes of this disease process. METHODS: A case-control survey was conducted using oesophageal tissue from 74 scleroderma cases and 74 age, race and sex-matched controls from our autopsy files. Histological evidence of oesophageal muscle atrophy was correlated with the degree of vascular changes, inflammatory infiltration, fibrosis, abnormalities of the myenteric plexus and reduction of interstitial cells of Cajal (ICC) using a predesigned semiquantitative descriptive method. RESULTS: Smooth-muscle atrophy was found in 94% of scleroderma cases, and in 5% of controls (p<0.001). Atrophy was evident in the circular smooth muscle in 93% of cases, and in the longitudinal smooth muscle in 66% of cases. Intimal proliferation of arterioles was found in 38% of cases and in 5% of controls (p<0.001), but was not associated with smooth-muscle atrophy (p = 0.29). Despite these vascular changes, there was no evidence of compromised perfusion, such as findings suggestive of acute ischaemic necroses. Minimal cellular infiltrates were seen in the myenteric plexus in 82% of cases and in 92% of controls (p = 0.091). ICC were found in fewer numbers in areas of atrophic smooth muscle compared with adjacent normal smooth muscle in selected scleroderma cases. CONCLUSION: The pathological findings of oesophageal lesions in scleroderma seem inconsistent with either an ischaemic or an inflammatory process. The loss of circular and longitudinal smooth muscle in the distal scleroderma oesophagus may represent loss of normal neural function followed by secondary tissue atrophy, or may be a primary smooth muscle lesion.

Increase in the rate of diploidy with maternal age in unfertilized in-vitro fertilization oocytes.

Human oocytes which failed to fertilize in vitro were fixed for cytogenetic analysis. Metaphase II chromosomes were identified in 286 oocytes, of which 233 were suitable for cytogenetic analysis. In all, 181 oocytes had a normal haploid karyotype (77.7%), while the remaining 52 were abnormal (28 aneuploid, 14 diploid and 10 tetraploid). The rate of aneuploidy did not increase with maternal age. However, the proportion of diploid oocytes increased significantly with advancing maternal age (P < 0.01), being particularly obvious in women aged > 35 years.

A simplified method for fixation of human and mouse preimplantation embryos which facilitates G-banding and karyotypic analysis.

A reliable method for the fixation of large numbers of preimplantation embryos was developed to allow application of routine G-banding methods to preimplantation stage embryos. The technique resulted in minimal loss of embryos and was suitable for both 1-cell mouse embryos and multi-pronuclear human embryos. Application of this method will increase the information gained from chromosome studies of embryonic development.

Contribution of reciprocal translocations to an understanding of chromosome displacement: inferences for studies of spatial order at metaphase.

Chromosome displacement was analysed in three different translocations. Two alternative hypotheses were examined: (1) Displacement is determined by the spatial ordering of chromosomes on the metaphase plate. (2) Displacement is a function of the gross physical property of chromosome size and does not reflect ordering. Predicted numbers of displacements were calculated for each chromosome for each of the two hypotheses and these were compared with the observed numbers of displacements using a chi 2 analysis. In the analysis for two of the three translocations, the first hypothesis was rejected whilst the alternative hypothesis was supported by all three analyses. It is concluded that chromosome displacement is a function of chromosome size and does not reflect spatial ordering at metaphase. Furthermore, it is suggested that many studies of apparent ordering at metaphase may merely reflect chromosome displacement. The analysis of displacement rates in all other chromosomes of the complement was undertaken in one of the translocation carriers. This showed no alteration of relative displacement rates.

Chromosome displacement and spindle tubule polymerization. 2. Effect of alterations of extracellular calcium on displacement parameters.

Human lymphocytes were grown for either the entire culture period of 72 h or the final 6 h of culture, in media with different concentrations of calcium. Cultures were harvested without colchicine or hypotonic treatment and 'displaced' chromosomes were analysed. Long or short term exposure to various concentrations of calcium gave similar results. The frequency of cells showing displacement and the median number of displaced chromosomes per cell were highly correlated with calcium concentration (r = 1). Only the smaller chromosomes, 13-22, showed a significant correlation for displacement rate and calcium concentration. Individual chromosomes showed a consistent response to calcium which appeared to be specific for each chromosome. Chromosome 17 was the most sensitive to calcium, but chromosomes 16, 19 and most C-group chromosomes showed no response. The acrocentric chromosomes 13, 14, 15, 21 and 22 all showed a modest but similar response. An hypothesis is advanced that these responses may be controlled by specific amounts of microtubular associated proteins and/or calmodulin in the spindle tubules attached to the different chromosomes.

Chromosome displacement and spindle tubule polymerization: 1. The effect of alterations in pH on displacement frequency.

In order to test whether 'chromosome displacement' (Ford and Lester, 1982) is related to spindle function, the phenomenon was examined under conditions known to alter spindle tubule polymerization, namely alterations in pH within the range 6.8-8.0. The following observations were made: (a) the frequency of cells showing chromosome displacement was not altered by variations in pH, but the number of displaced chromosomes per cell was markedly changed. Minimum numbers of chromosomes were displaced at pH 7.6. (b) For any chromosome, the extent of the response to pH change, was positively correlated with the basal displacement rate for that chromosome. (c) Chromosomes which have a 'stable' evolutionary history have a more predictable response to pH than those which have an 'unstable' history. Since displacement is significantly influenced by pH, it is concluded that the phenomenon is related to spindle structure.

Premature ovarian failure and ovarian dysgenesis associated with balanced and unbalanced X-6 translocations, respectively: implications for the investigation of ovarian failure.

This study reports the effect of an inherited (X;6) translocation which has not previously been described. The proband was intellectually delayed and had ovarian dysgenesis. Karyotyping revealed an unbalanced karyotype: 46,X,der(X)t(X;6)(q22; p11.2)*. Her mother was shown to be a carrier of an apparently balanced translocation between the X chromosome and chromosome 6: 46,X,t(X;6)(q22;p11.2). This finding in the mother raises to 7 the number of cases reported which involve a break within the X chromosome 'critical region', at band Xq22, without causing ovarian dysgenesis, although it was associated with premature ovarian failure. These cases aim to highlight to clinical specialists the range of gonadal and other phenotypic anomalies (apart from those associated with Turner syndrome) which can occur due to partial deletions of the X chromosome. These findings have implications for the investigation of both ovarian dysgenesis and premature ovarian failure.

A simple and rapid method for the identification of cycling cells in freshly excised tumours.

Cytogenetic studies of fresh solid tumours are hampered by the small numbers of poor quality metaphases which are obtained through direct preparations. The proportion of cycling cells in freshly excised tumours have been identified by measuring the incorporation of the nucleoside analogue 5-bromo-2'deoxyuridine (BrUdR) into DNA. We have developed an immunochemical method using a mouse monoclonal antibody directed against BrUdR which stains nuclei of cells which have incorporated this nucleoside. Using this method, optimal culture conditions and harvest times may be identified, to provide greater numbers of high quality metaphases, suitable for karyotyping solid tumours.

Use of pulsed electromagnetic fields in treatment of loosened cemented hip prostheses. A double-blind trial.

A double-blind trial of pulsed electromagnetic fields (PEMFs) for loosened cemented hip prostheses was conducted at two centers. Of the 40 patients who enrolled, 37 met entry criteria and were available for analysis. All patients completed six months of treatment (either active or control units). Success was determined clinically by a Harris hip score greater than or equal to 80 points (or an increase of ten points if initially greater than or equal to 70 points). Ten of the 19 active units were successes (53%), whereas two of the 18 controls (11%) exhibited a placebo effect, a statistically significant and clinically relevant result. A 60% relapse rate among the active successes was seen at 14 months poststimulation, and despite maintenance therapy of one hour per day, the relapse rate increased to 90% at three years. These data suggest that for loosened cemented hip prostheses, use of PEMFs is a treatment option only to delay revision hip surgery.