Multiantigen print immunoassay (MAPIA) for the diagnosis of neurocysticercosis: a single-center diagnostic optimization and accuracy study in Lima, Peru.

Neurocysticercosis (NCC) is the most common helminthic infection of the human central nervous system. The antibody detection assay of choice is the enzyme-linked immunoelectrotransfer blot assay using lentil-lectin purified parasite antigens (LLGP-EITB, Western blot), an immunoassay with exceptional performance in clinical samples. However, its use is mainly restricted to a few research laboratories because the assay is labor-intensive and requires sophisticated equipment, expertise, and large amounts of parasite material for preparation of reagents. We report a new immunoprint assay (MAPIA) that overcomes most of these barriers. We initially compared the performance of five different antigen combinations in a subset of defined samples in the MAPIA format. After selecting the best-performing assay format (a combination of rGP50 + rT24H + sTs14 antigens), 148 archived serum samples were tested, including 40 from individuals with parenchymal NCC, 40 with subarachnoid NCC, and 68 healthy controls with no evidence of neurologic disease. MAPIA using three antigens (rGP50 + rT24H + sTs14) was highly sensitive and specific for detecting antibodies in NCC. It detected 39 out of 40 (97.5%) parenchymal NCC cases and 40/40 (100%) subarachnoid cases and was negative in 67 out of 68 (98.53%) negative samples. MAPIA using three recombinant and synthetic antigens is a simple and economical tool with a performance equivalent to the LLGP-EITB assay for the detection of specific antibodies to NCC. The MAPIA overcomes existing barriers to adoption of the EITG LLGP and is a candidate for worldwide use.

Neurocysticercosis. A frequent cause of seizures, epilepsy, and other neurological morbidity in most of the world.

Neurocysticercosis is endemic in most of the world and in endemic areas it accounts for approximately 30% of cases of epilepsy. Appropriate diagnosis and management of neurocysticercosis requires understanding the diverse presentations of the disease since these will vary in regards to clinical manifestation, sensitivity of diagnostic tests, and most importantly, therapeutic approach. This review attempts to familiarize tropical neurology practitioners with the diverse types of neurocysticercosis and the more appropriate management approaches for each.

Evaluación de películas comestibles de almidón de yuca y proteína aislada de soya en la conservación de fresas / Evaluation of edible films of cassava starch and isolated soy protein in the preservation of strawberries

A partir de diferentes proporciones de almidón de yuca y proteína aislada de soya, se elaboraron películas comestibles por el método de casting, las cuales se caracterizaron a través de pruebas mecánicas, térmicas y morfológicas. Se evaluó el desempeño de las películas como recubrimiento sobre las fresas variedad ventana mediante la determinación de propiedades sensoriales y fisicoquímicas: color, apariencia, aroma, sabor, textura, pH, acidez titulable y sólidos solubles totales a temperaturas ambiente y de refrigeración. En las formulaciones que contenían proteína se observó una mayor elasticidad, eventos térmicos a temperatura más baja comparados con los de las mezclas, una superficie más homogénea, permitiendo mejorar algunas propiedades del fruto durante su almacenamiento como la pérdida de peso. Las propiedades sensoriales se evaluaron utilizando las pruebas de Kruskal Wallis y Friedman, obteniendo en la primera diferencias no significativas (p>0.05) entre los tratamientos evaluados y diferencias significativas en la segunda, observando un desempeño favorable en los recubrimientos comestibles.

Afrontamiento primario con elásticos vasculares en el manejo del cierre progresivo de fasciotomías / Vessel loops for primary closure of fasciotomies

Background: Compartment syndrome of the extremity may occur after severe trauma with vascular lesions secondary to fractures, crushes or gunshots. To prevent it a fasciotomy must be done. Aim To report the use vessel loop shoelace technique for the progressive closure of the fasciotomy. Material and methods: Descnptive study of 24 patients aged 26 +/- 9 years (21 males) that required fasciotomy to prevent compartment syndromes. The fasciotomy wound was closed progressively using vessel loops anchored to the skin with staples or sutures, which were tightened progressively, according to the evolution. Results: The studied patients required a total of 56 fasciotomies. In all patients a complete or near complete closure of the wound was achieved. The mean closure time was 9.5 +/- 3.3 days. Mean hospital stay was 12.3 +/- 4.3 days. Conclusions: Vessel loop shoelace technique is effective for fasciotomy wound closure.

Frente al cada vez más frecuente manejo del trauma vascular en extremidades, debido a lesiones de distinta etiología, ya sea por accidentes de tránsito de alta velocidad, caídas de altura, o las crecientes tasas de lesiones en la vida civil por arma blanca y por arma de fuego, es que nos vemos enfrentados a múltiples tipos de lesiones vasculares que comprometen arterias y venas, asociados a extenso compromiso de partes blandas o a reintervenciones en períodos posteriores a las 6 hrs post trauma. De este modo es que el manejo asociado de la fasciotomía como método de prevención o tratamiento del síndrome compartamental es fundamental. Se presentan un total de 24 casos en los que se efectuaron 56 fasciotomías primarias o secundarias como profilaxis o tratamiento del síndrome compartamental, en las que se utilizaron elásticos vasculares para el cierre progresivo del defecto cutáneo de las fasciotomías, para disminuir el tiempo de evolución y así evitar el uso de injertos de piel en el cierre de los defectos.

The Ras oncogene signals centrosome amplification in mammary epithelial cells through cyclin D1/Cdk4 and Nek2.

Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-Ras(G12D) and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-Ras(G12D), it was detected only in premalignant mammary lesions expressing K-Ras(G12D). CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-Ras(G12V) abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA.

Transitional sublethal and lethal effects of insecticides after dermal exposures to five economic species of wireworms (Coleoptera: Elateridae).

During an insecticide toxicity study involving field-collected dusky wireworm, Agriotes obscurus (L.) (Coleoptera: Elateridae), wireworms exposed dermally to six classes of insecticides exhibited characteristic transitional symptoms of toxicity. These symptoms, collectively termed "morbidity," were categorized as "writhing," "leg and mouthpart movements," or "mouthpart-only body movements." These symptoms could persist for long periods, depending on insecticide and dose, with morbid wireworms ultimately recovering or dying. Additional LC50 and LD50 toxicity studies showed that these stages of morbidity also occurred in four other wireworm species, notably Agriotes sputator (L.), Limonius canus LeConte, Ctenicera pruinina (Horn), and Ctenicera destructor (Brown). In addition, all species exposed dermally to clothianidin moved in significant numbers to the surface of soil in posttreatment holding cups. This movement was not observed when these species were exposed to chlorpyrifos or the control solvent. These findings suggest that toxicity trials involving wireworms should include observations on morbidity, and the duration of trials should continue until symptoms of morbidity cease. The long-term morbidity and potential recovery or death of wireworms exposed to certain insecticides has implications for how laboratory and field studies can be better designed and interpreted in the future.

Mortality of a wireworm, Agriotes obscurus (Coleoptera: Elateridae), after topical application of various insecticides.

Ten insecticides representing seven chemical groups were applied at various concentrations topically by using a Potter Spray Tower to evaluate their relative toxicities on the European wireworm Agriotes obscurus L. (Coleoptera: Elateridae). Wireworms were stored at 15 degrees C after exposure to organophosphate (OP) (chlorpyrifos, diazinon), pyrethroid (tefluthrin), thianicotinoid (thiamethoxam, clothianidin), chloronicotinoid (imidacloprid, acetamiprid), phenyl pyrazole (fipronil), organochlorine (lindane), and spinosyn (spinosad) insecticides, and their postapplication health was evaluated weekly for up to 301 d. LC50, LC90, LT50, and LT90 values were calculated for each chemical except acetamiprid, and compared with those of lindane, clothianidin, and chlorpyrifos. Wireworms exposed to OPs died or recovered more quickly (LT50 < 20 d, LT90 < 50 d), than those exposed to all other insecticides tested except tefluthrin (LT50 = 25.5 d, LT90 = 66.5 d). Wireworms exposed to sublethal concentrations of all neonicotinoids quickly became moribund after application but made a full recovery. Wireworms exposed to fipronil at concentrations near the LC90 value showed no intoxication symptoms for up to 35 d, and they did not recover after symptoms developed. For each chemical, increasing the concentration increased the time required for wireworms to recover but decreased the time required to kill wireworms. Fipronil was highly toxic to wireworms (LC50 = 0.0001%), but acetamiprid (LC50 = 1.82%), imidacloprid (LC50 = 0.83%), tefluthrin (LC50 = 0.23%), diazinon (LC50 = 0.54%), and spinosad (LC50 = 0.51%) were not. The toxicity of both clothianidin (LC50 = 0.07%) and thiamethoxam (LC50 = 0.17%) were similar to those oflindane (LC50 = 0.06%) and chlorpyrifos (LC50 = 0.10%).

The E2F1-3 transcription factors are essential for cellular proliferation.

The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.

Failure of one-day praziquantel treatment in patients with multiple neurocysticercosis lesions.

A recently described one-day regimen of praziquantel (PZQ) therapy for neurocysticercosis (NCC), three doses of 25 mg/k given at 2 h intervals, was applied in eight patients with viable NCC cysts without any evidence of inflammation. Resolution of lesions in computed tomography (CT) was observed in all five patients with a single cyst, whereas all cysts survived in three patients with multiple brain parasites. One-day praziquantel is a good regimen for patients with a single viable brain cysticercus but is poorly effective for multiple cysts.

Myc requires distinct E2F activities to induce S phase and apoptosis.

Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.

Conditional apoptosis induced by oncogenic ras in thyroid cells.

Mutations of ras are tumor-initiating events for many cell types, including thyrocytes. To explore early consequences after oncogenic Ras activation, we developed a doxycycline-inducible expression system in rat thyroid PCCL3 cells. Beginning 3-4 days after H-Ras(v12) expression, cells underwent apoptosis. The H-Ras(v12) effects on apoptosis were decreased by a mitogen-activated protein kinase kinase (MEK1) inhibitor and recapitulated by doxycycline-inducible expression of an activated MEK1 mutant (MEK1(S217E/S221E)). As reported elsewhere, acute expression of H-Ras(v12) also induces mitotic defects in PCCL3 cells through ERK (extracellular ligand-regulated kinase) activation, suggesting that apoptosis may be secondary to DNA damage. However, acute activation of SAPK/JNK (stress-activated protein kinase/Jun N-terminal kinase) through acute expression of Rac1(v12) also triggered apoptosis, without inducing large-scale genomic abnormalities. H-Ras(v12)-induced apoptosis was dependent on concomitant activation of cAMP by either TSH or forskolin, in a protein kinase A-independent manner. Thus, coactivation of cAMP-dependent pathways and ERK or JNK (either through H-Ras(v12), Rac1(v12), or MEK1(S217E/S221E)) is inconsistent with cell survival. The fate of thyrocytes within the first cell cycles after expression of oncogenic Ras is dependent on ambient TSH levels. If both cAMP and Ras signaling are simultaneously activated, most cells will die. Those that survive will eventually lose TSH responsiveness and/or inactivate the apoptotic cascade through secondary events, thus enabling clonal expansion.

The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway.

Activating mutations of RAS are thought to be early events in the evolution of thyroid follicular neoplasms. We used a doxycycline-inducible expression system to explore the acute effects of H-RAS12 on genomic stability in thyroid PCCL3 cells. At 2-3 days (first or second cell cycle) there was a significant increase in the frequency of micronucleation. Treatment of cells with YVAD-CHO inhibited RAS-induced apoptosis, but had no effect on micronucleation. The effects of H-RAS(V12) were mediated by activation of MAPK, as treatment with PD98059 at concentrations verified to selectively inhibit MEK1 reduced the frequency of prevalence of cells with micronuclei. In addition, doxycycline-inducible expression of a constitutively active MEK1, but not of a mutant RAC1, mimicked the effects of H-RAS(V12). The effects of H-RAS(V12) on genome destabilization were apparent even though the sequence of p53 in PCCL3 cells was confirmed to be wild-type. Acute activation of H-RAS(V12) evoked a proportional increase in both CREST negative and CREST positive micronuclei, indicating that both clastogenic and aneugenic effects were involved. H-RAS(V12) and activated MEK1 also induced centrosome amplification, and chromosome misalignment. Evidence that acute expression of constitutively activated RAS destabilizes the genome of PCCL3 cells is consistent with a mode of tumor initiation in which this oncogene promotes phenotypic progression by predisposing to large scale genomic abnormalities.

MAPK mediates RAS-induced chromosome instability.

The generation of micronuclei is a reflection of DNA damage, defective mitosis, and loss of genetic material. The involvement of the MAPK pathway in mediating v-ras-induced micronuclei in NIH 3T3 cells was examined by inhibiting MAPK activation. Conversely, the MAPK pathway was constitutively activated by infecting cells with a v-mos retrovirus. Micronucleus formation was inhibited by the MAPK kinase inhibitors PD98059 and U0126, but not by wortmannin, an inhibitor of the Ras/phosphatidylinositol 3-kinase pathway. Transduction of cells with v-mos resulted in an increase in micronucleus formation, also consistent with the involvement of the MAPK pathway. Staining with the anti-centromeric CREST antibody revealed that instability induced by constitutive activation of MAPK is due predominantly to aberrant mitotic segregation, since most of the micronuclei were CREST-positive, reflective of lost chromosomes. A significant fraction of the micronuclei were CREST-negative, reflective of lost acentric chromosome fragments. Some of the instability observed was due to mitotic events, consistent with the increased formation of bi-nucleated cells, which result from perturbations of the mitotic spindle and failure to undergo cytokinesis. This chromosome instability, therefore, is a consequence of mitotic aberrations, mediated by the MAPK pathway, including centrosome amplification and formation of mitotic chromosome bridges.

Management implications of evaluating the N2 and N3 neck after organ preservation therapy.

OBJECTIVES/HYPOTHESIS: To determine if metastatic squamous cell carcinoma with proliferative potential persists in N2 and N3 necks after conventional radiation. STUDY DESIGN: Retrospective case series. MATERIALS AND METHODS: We identified 17 patients from our head and neck tumor database who underwent organ-preserving radiotherapy for primary aerodigestive squamous cell cancer and N2-3 regional metastasis. Archival tissue from these 17 neck specimens was evaluated for routine histopathologic evidence of tumor, as well as immunohistochemically for cytokeratin and Ki-67 activity. An assay for apoptosis was also performed on 10 of the specimens. RESULTS: Routine H&E evaluation suggested metastatic cancer in 11 of 17 irradiated neck specimens. Cytokeratin immunostaining confirmed squamous cell carcinoma in these 11 necks as well as 1 additional specimen that had tested H&E negative. Ki-67 staining demonstrated proliferating tumor in 3 of 17 necks. The apoptosis assay confirmed regions of apoptosis in all of the specimens analyzed. CONCLUSIONS: The discovery of proliferating cancer cells in 3 of 17 irradiated specimens (18%) supports the practice of planned neck dissection after primary radiotherapy for patients with pretherapeutic N2+ metastatic disease.

Alloantigen gene therapy for squamous cell carcinoma of the head and neck: results of a phase-1 trial.

OBJECTIVE: To determine the safety and efficacy of an immunogenic gene therapy using a drug designed to produce expression of a foreign class I major histocompatibility complex protein in patients with head and neck cancer. DESIGN: Phase 1 prospective clinical trial. SETTING: Academic medical setting. PATIENTS: Nine patients with advanced head and neck squamous cell carcinoma who had failed conventional therapy and did not express HLA-B7, a class I major histocompatibility complex protein. INTERVENTION: Patients were treated with Allovectin-7 (Vical Inc, San Diego, Calif) by direct intratumoral injection. Allovectin-7 contains a plasmid complementary DNA complexed with a cationic lipid, which results in expression of HLA-B7. MAIN OUTCOME MEASURES: Patients were assessed for any toxic effects and for any change in tumor volume. Biopsy specimens obtained before and after therapy were evaluated by immunohistochemistry to detect HLA-B7 expression and with the terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay to detect any induction of apoptosis. RESULTS: There were no toxic effects of the gene therapy. In 4 of these 9 patients there was a partial response to treatment, evidenced by a gradual reduction in tumor size. One patient has remained alive for more than 17 months since commencing treatment, with no clinical evidence of disease but with persistent histological evidence of cancer. Analysis of the biopsy specimens from 2 of the patients who responded to therapy demonstrated HLA-B7 expression. The TUNEL assay demonstrated extensive apoptosis in both of these patients, suggesting that this may be the mechanism of tumor reduction. CONCLUSIONS: These data demonstrate the potential efficacy and lack of toxicity of this form of alloantigen gene therapy. A multi-institutional study has been initiated to expand on these findings.

Interleukin-3 increases the incidence of 5-azacytidine-induced thymic lymphomas in pBOR-Il-3 mice.

Interleukin-3 (Il-3) is a glycoprotein produced by a CD4+CD8- subpopulation of T-lymphocytes. Il-3 has been associated with the proliferation of bone marrow stem cells and their differentiation to granulocytes, macrophages, basophil/mast cells, megakaryocytes, erythroid cells, and neutrophils. The pBOR-Il-3 transgenic mice were developed by pronuclear microinjection to study how chemical insults modulate transcription of the Il-3 gene driven by a long-terminal repeat (LTR) of an endogenous retrovirus and to determine the biological consequences of interleukin-3 expression. We injected 5-azacytidine, a demethylating agent, to increase the LTR-driven expression of Il-3. Upon 5-azacytidine treatment, both the pBOR-Il-3 and the FVB/N nontransgenic controls developed thymic lymphomas. The pBOR-Il-3 mice developed thymic lymphomas at a higher frequency than the FVB/N mice. The thymic lymphoma cells were of a T-cell origin, as determined by T-cell receptor gene rearrangement analysis, and, in most cases, were of monoclonal origin. According to flow cytometric analysis of CD3, CD4, and CD8 cell surface markers, the thymic lymphoma cells did not lose their ability to differentiate, but the differentiation process was aberrant. Flow cytometric analyses also revealed that in pBOR-Il-3 mice the thymic lymphomas are mostly of a CD8+CD4- origin, whereas in the FVB/N group, the predominant type of thymic lymphoma is of a CD4+CD8- origin.

Neuronal changes induced by chronic toluene exposure in the cat.

Chronic toluene inhalation provokes significant deleterious neurological effects in young glue sniffers and exposed workers. We have developed a chronic toluene inhalation model in the cat to address this issue. Neuronal changes using Loyez and acid fuchsinegallocianine stainings were studied at prefrontal cortex, cerebellun and hippocampus. All this structures showed varying degrees of neuronal degeneration to necrosis. Even if injury signs were widespread, the neuronal layers weren't equally affected and there were clear differences in injury severity. In the prefrontal cortex, injured neurons were observed in layers II, III and V/VI extending over several gyri. Lesions were time related, as was more clearly observed in Purkinje cells. In dorsal hippocampus alterations were particulary severe in CA1 and CA3. In ventral hippocampus damaged neurons were scarce and located mainly in CA2. The possible relation of this findings with behavioral changes observed during chronic toluene inhalation are noted.

Glutamate, GABA, calbindin-D28k and parvalbumin immunoreactivity in the pulvinar-lateralis posterior complex of the cat: relation to the projection to the Clare-Bishop area.

Neurons of the pulvinar-lateralis posterior complex (Pul-LP) containing glutamate (Glu) and GABA, as presumed neurotransmitters, and calbindin- D28k (calbindin) and parvalbumin (PV), as Ca-binding proteins, were identified in the cat by using immunohistochemical methods. In vibratome sections, neurons immunoreactive (IR) to each of the four antibodies were observed throughout the Pul-LP. In semithin sections, GABA-IR neurons were also PV-IR but not calbindin-IR and some of them also co-localized Glu. The Glu-IR neurons which were negative for GABA co-localized calbindin but not PV. The neurons of the Pul-LP projecting to the Clare-Bishop area (CB) in the suprasylvian gyrus were identified with a retrogradely transported tracer and the sections were then immunostained for Glu, GABA, calbindin and PV. Only Glu- and calbindin-IR neurons were retrogradely labeled. These results show that, if calbindin and PV have a Ca-binding role, the presumably excitatory Glu-IR neurons projecting to the CB are use calbindin whereas the presumably inhibitory GABA-IR neurons are intrinsic and use PV. This relationship implies that these proteins probably have other roles specifically related to the kind of agonist to be released at the neuron.

Successful treatment of Trypanosoma cruzi encephalitis in a patient with hemophilia and AIDS.

Although the frequency of infection with the human immunodeficiency virus (HIV) is increasing dramatically in areas where Trypanosoma cruzi is endemic, trypanosomiasis has been rarely reported in persons with HIV infection or AIDS. Persons with hemophilia who receive multiple blood product transfusions from blood banks with little or no screening for infectious agents are at particularly high risk for infections with both HIV and T. cruzi. We describe the case of a person with hemophilia who was infected by blood transfusion with HIV and T. cruzi and in whom a multifocal, necrotic trypanosomal encephalitis was demonstrated by brain biopsy and electron microscopy. Treatment with benznidazole followed by that with itraconazole and fluconazole was associated with significant clinical and radiographic improvement.