Unraveling the gut microbiota of Mexican pinnipeds: the dominance of life histories over phylogeny.

Studying how phylogeny influences the composition and functions of microbiotas within animal hosts is essential for gaining insights into the connection between genetics, ecology, and health in the animal kingdom. However, due to limited comprehensive studies, this influence remains unclear for many wild mammals, including Mexican pinnipeds. We employed 16S rRNA gene deep-sequencing to investigate the impact of phylogeny on the gut microbiota of four pinniped species inhabiting Mexican shores: the Pacific harbor seal (Phoca vitulina richardii), the northern elephant seal (Mirounga angustirostris), the California sea lion (Zalophus californianus), and the Guadalupe fur seal (Arctocephalus philippii townsendi). Our results indicated that factors such as diets and shared life histories exerted more influence on microbiota composition than phylogeny alone. Notably, otariid species sharing similar life histories displayed greater microbiota similarity than phocids, which have distinct life histories and fewer microbiota similarities. Furthermore, harbor seals have more microbial similarities with the two otariid species than with elephant seals. Of particular concern, we observed a higher abundance of potentially pathogenic bacteria (e.g., Photobacterium damselae and Clostridium perfringens) in harbor seals and Guadalupe fur seals compared to other pinnipeds. This finding could pose health threats to these species and nearby human populations.IMPORTANCEPinnipeds in Mexico host microbial communities that remain understudied. While several factors can influence microbiota composition, the role of phylogenetic relationships among these pinnipeds remains unclear due to limited knowledge of the microbiota in certain species. This study aimed to fill this gap by characterizing the composition and function of the gut microbiota in the four pinniped species that occur in Mexico. Our analysis reveals that shared diets and life histories contribute to similarities in the composition of gut microbial communities. This study also highlights the potential differences in the metabolic capabilities and adaptations within the gut microbiota of pinnipeds. Understanding how phylogeny impacts microbial communities enhances our insights into the evolutionary dynamics of marine mammals.

Metabarcoding Used for the First Time to Identify Prey of Wild Totoaba macdonaldi.

Totoaba macdonaldi is an endangered endemic fish of the Gulf of California. Overexploitation resulted in the Mexican government banning the fishing of this species in 1975, and it being listed as endangered. However, the species is still subject to illegal fishing. Despite its conservation status, little is known about totoaba biology. The present study aimed to implement, for the first time, a metabarcoding protocol to describe the totoaba diet. Four wild totoaba individuals, seized by Mexican law enforcement agents, were dissected, and their stomach contents were collected. Three representative amplicon libraries were generated for cephalopods, chordates, and eukaryotes. After sequencing, 18 different taxa were identified, of which 11 species were recognized as prey. The totoaba were found to have consumed Pacific anchovy (Cetengraulis mysticetus), flathead grey mullet (Mugil cephalus), bigeye croaker (Micropogonias megalops), northern anchovy (Engraulis mordax), ocean whitefish (Caulolatilus princeps), milkfish (Chanos chanos), and Pacific sardine (Sardinops sagax). Members of the Euphausiidae family (krill) were also identified. This study identified up to four times more species in much fewer samples than previous studies based on morphological recognition, thus confirming metabarcoding as an effective method for studying the feeding habits of this species and one providing the tools required for further analysis of the totoaba diet.

[Hemicerebellitis due to chikungunya associated with refractory status epilepticus in the paediatric age]. / Hemicerebelitis por chikungunya asociado a estado epiléptico refractario en edad pediátrica.

TITLE: Hemicerebelitis por chikungunya asociado a estado epiléptico refractario en edad pediátrica.

Cation and anion effect on the biodegradability and toxicity of imidazolium- and choline-based ionic liquids.

This work studies the effect of the cation and anion on the biodegradability and inhibition of imidazolium- and choline-based ionic liquids (ILs) using activated sludge. Six commercial ILs, formed by combination of 1-Butyl-3-methylimidazolium (Bmim+) and N,N,N-trimethylethanolammonium (Choline+) cations and chloride (Cl-), acetate (Ac-) and bis(trifluoromethanesulfonyl)imide (NTf2-) anions were evaluated, all representative counter-ions with markedly different toxicity and biodegradability. Inherent and fast biodegradability tests were used to evaluate both the microorganism inhibition and the IL biodegradability. In addition, the ecotoxicological response (EC50) of the ILs was studied using activated sludge and Vibrio fischeri (Microtox® test). Bmim+ and NTf2- can be considered as non-biodegradable, whereas aerobic microorganisms easily degraded Choline+ and Ac-. The biodegradation pattern of each cation/anion is nearly unaffected by counter-ion nature. Moreover, concentrations of CholineNTf2 higher than 50 mg/L caused a partial inhibition on microbial activity, in good concordance with its low EC50 (54 mg/L) measured by respiration inhibition test, which alerts on the negative environmental impact of NTf2-containing ILs on the performance of sewage treatment plants.

Transcriptomic response of the Crassostrea virginica gonad after exposure to a water-accommodation fraction of hydrocarbons and the potential implications in reproduction.

The Crassostrea virginica oyster has biological and economic importance in the Gulf of Mexico, an area with a high extraction and production of hydrocarbons. Exposure to hydrocarbons affects the reproductive processes in bivalves. In C. virginica, the effect of hydrocarbons on the gonad of the undifferentiated organism has not been evaluated to determine the possible damage during the maturation process. To evaluate this effect, RNA-seq data was generated from C. virginica gonads exposed to a 200 µg/L of hydrocarbons at different exposure times (7, 14 and 21 days) and a control treatment (without hydrocarbons). The analysis of the gonad transcriptome showed the negative effect of hydrocarbons on maturation, with a sub-expression of 22 genes involved in different stages of this process. Additionally, genes in the immune system were down-regulated, which may indicate that exposure to hydrocarbons causes immunosuppression in bivalves. A group of oxidative stress genes was also reduced. These data contribute to a better understanding of the effect of hydrocarbons on the reproductive process in bivalves and, at the same time, allow us to identify possible biomarkers associated with hydrocarbon contamination in the gonad of C. virginica.

Assessment the ecotoxicity and inhibition of imidazolium ionic liquids by respiration inhibition assays.

The ecotoxicity and inhibition of 12 imidazolium ionic liquids (ILs) with alkyl chain from C4 to C10 and chloride (Cl-), tetrafluoroborate (BF4-) and bis(trifluoromethanesulfonyl)imide (NTf2-) anions have been studied by means of respiration inhibition assays using activated sludge collected from a wastewater treatment plant. This test represents an alternative easy, economic and quick way to evaluate the true impact of ILs on activated sludge-based wastewater treatment. For comparison purposes, the EC50 values were also determined by the Microtox test (Vibrio fischeri). It was observed that this widely used microbial test overestimates the effect of the ILs on biological wastewater treatment facilities, especially in the case of ILs with lower ecotoxicity. The results of the biological tests showed that the alkyl chain length plays a crucial role in the ecotoxicity of ILs. A significant increase of the toxicity with the length of the n-alkyl chain was found. Regarding to the impact of the anion, the ecotoxicity measured by respiration inhibition assays follows the order NTf2- > Cl- > BF4-, being the anion effect higher as decreasing the length of cation alkyl chain. According to the hazard substances ranking for aquatic organisms (Passino and Smith, 1987), imidazolium ILs with C4 alkyl chain can be classified as "practically harmless" compounds whereas those with alkyl chains C8 or C10 correspond to "highly toxic" species.

Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1µM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins.

Nivel de actividad física según GPAQ en mujeres embarazadas y postparto que asisten a un centro de salud familiar / Physical activity levels using the GPAQ in women during pregnancy and postpartum

Antecedentes: Durante el embarazo y postparto las mujeres disminuyen el nivel de actividad física (AF). Objetivo: Determinar el nivel de AF y conducta sedentaria en mujeres embarazadas y postparto. Métodos: Participaron mujeres embarazadas y postparto entre 18 y 40 años de edad de un Centro de Salud Familiar. La AF y conducta sedentaria se midió utilizando el Global Physical Activity Questionnaire. Resultados: Se analizó la información de 47 mujeres embarazadas y 12 en postparto. La edad P50=25 años (rango: 18-40 años). El 61 por ciento presentó exceso de peso. El tiempo de AF diaria fue P50=154 (rango: 0-960 min/día) en embarazadas y P50=190 (rango: 24-411 min/día) en postparto. La AF en la dimensión tiempo libre en embarazadas fue P50=0 (rango: 0-540 min/día) y P50=3 (rango: 0-86 min/día) en el grupo de postparto. La conducta sedentaria para todo el grupo fue P50=180 (rango: 0-720 min/día). El 20,3 por ciento de las mujeres clasifica como suficientemente activo según criterio OMS, al corregir la AF reportada por GPAQ según IMC y nivel educacional. No hubo asociación entre las variables edad, estado nutricional, nivel educacional e ingreso familiar con el nivel de AF reportado. Conclusión: La AF de tiempo libre es una dimensión susceptible a intervenciones de promoción de la AF. El nivel de AF reportado por el grupo es mayor a resultados de estudios similares en otros países. Estudios de validación en esta población son necesarios para determinar la posible sobreestimación de los resultados por un sesgo de medición.

Background: During pregnancy and postpartum, women decrease physical activity (PA) levels despite the health benefits reported in the literature for women and the fetus. Aims: To determine PA levels and sedentary behaviour in women during pregnancy and postpartum. Methods: Pregnant and postpartum women between 18 and 40 years old attending to primary care center participated. Sedentary behaviour and PA were measured using the Global Physical Activity Questionnaire (GPAQ). Results: Information was collected from 47 pregnant and 12 postpartum women. Age was P50=25 (range: 18-40 years). 61 percent were overweight. Daily PA time was P50=154 (range: 0-960 min/day) for pregnant women and P50=190 (range: 24-411 min/day) during postpartum. Leisure time PA was P50=0 (range: 0-540 min/day) for pregnant women and P50=3 (range: 0-86 min/day) during postpartum. Sedentary behaviour was P50=180 min/day (range: 0-720 min/day) for all women. According to WHO criteria, only 20.3 percent of women are physically active, adjusting by BMI and educational level the reported PA in GPAQ. There was no association between the variables age, nutritional status, educational level, family income and reported level of PA. Conclusion: Leisure time PA could be susceptible to PA promotion interventions. The reported level of PA is higher than those from similar studies in other countries. Validation studies are needed to determine possible overestimation of the results due to measurement bias.

The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection.

African swine fever virus (ASFV) infection induces apoptosis in the infected cell; however, the consequences of this activation on virus replication have not been defined. In order to identify the role of apoptosis in ASFV infection, we analyzed caspase induction during the infection and the impact of caspase inhibition on viral production. Caspases 3, 9 and 12 were activated from 16 h post-infection, but not caspase 8. Indeed, caspase 3 activation during the early stages of the infection appeared to be crucial for efficient virus exit. In addition, the inhibition of membrane blebbing reduced the release of virus particles from the cell. ASFV uses the endoplasmic reticulum (ER) as a site of replication and this process can trigger ER stress and the unfolded protein response (UPR) of the host cell. In addition to caspase 12 activation, indicators of ER stress include the upregulation of the chaperones calnexin and calreticulin upon virus infection. Moreover, ASFV induces transcription factor 6 signaling pathway of the UPR, but not the protein kinase-like ER kinase or the inositol-requiring enzyme 1 pathways. Thus, the capacity of ASFV to regulate the UPR may prevent early apoptosis and ensure viral replication.

Myalgic encephalomyelitis: International Consensus Criteria.

The label 'chronic fatigue syndrome' (CFS) has persisted for many years because of the lack of knowledge of the aetiological agents and the disease process. In view of more recent research and clinical experience that strongly point to widespread inflammation and multisystemic neuropathology, it is more appropriate and correct to use the term 'myalgic encephalomyelitis' (ME) because it indicates an underlying pathophysiology. It is also consistent with the neurological classification of ME in the World Health Organization's International Classification of Diseases (ICD G93.3). Consequently, an International Consensus Panel consisting of clinicians, researchers, teaching faculty and an independent patient advocate was formed with the purpose of developing criteria based on current knowledge. Thirteen countries and a wide range of specialties were represented. Collectively, members have approximately 400 years of both clinical and teaching experience, authored hundreds of peer-reviewed publications, diagnosed or treated approximately 50 000 patients with ME, and several members coauthored previous criteria. The expertise and experience of the panel members as well as PubMed and other medical sources were utilized in a progression of suggestions/drafts/reviews/revisions. The authors, free of any sponsoring organization, achieved 100% consensus through a Delphi-type process. The scope of this paper is limited to criteria of ME and their application. Accordingly, the criteria reflect the complex symptomatology. Operational notes enhance clarity and specificity by providing guidance in the expression and interpretation of symptoms. Clinical and research application guidelines promote optimal recognition of ME by primary physicians and other healthcare providers, improve the consistency of diagnoses in adult and paediatric patients internationally and facilitate clearer identification of patients for research studies.

The effect of sildenafil citrate (Viagra) on cerebral blood flow in patients with cerebrovascular risk factors.

OBJECTIVES: Sildenafil citrate is widely used for erectile dysfunction. The present study examined the short-term effects of sildenafil administration in individuals with cerebrovascular risk factors, including patients with a history of stroke. MATERIALS AND METHODS: Twenty-five consecutive male patients with erectile dysfunction and vascular risk factors were included in the study. A perfusion brain SPECT study was performed at baseline and 1 h after the oral administration of sildenafil. RESULTS: Associations between any of the risk factors and the perfusion scores were not detected, with the exception of stroke. Stroke patients showed significantly more areas with diminished perfusion after sildenafil administration compared to baseline. CONCLUSIONS: In patients with diabetes or hypertension, a dose of 50 mg sildenafil does not appear to produce detrimental effects on cerebral blood flow. However, patients with a history of stroke may be at increased risk of hemodynamic impairment after the use of sildenafil.

Chimeric calicivirus-like particles elicit protective anti-viral cytotoxic responses without adjuvant.

We have analyzed the potential of virus-like particles (VLPs) from rabbit hemorrhagic disease virus (RHDV) as a delivery system for foreign T cell epitopes. To accomplish this goal, we generated chimeric RHDV-VLPs incorporating a CD8(+) T cell epitope (SIINFEKL) derived from chicken ovalbumin (OVA). The OVA epitope was inserted in the capsid protein (VP60) of RHDV at two different locations: 1) the N-terminus, predicted to be facing to the inner core of the VLPs, and 2) a novel insertion site predicted to be located within an exposed loop. Both constructions correctly assembled into VLPs. In vitro, the chimeric VLPs activated dendritic cells for TNF-alpha secretion and they were processed and presented to specific T cells. In vivo, mice immunized with the chimeric VLPs without adjuvant were able to induce specific cellular responses mediated by cytotoxic and memory T cells. More importantly, immunization with chimeric VLPs was able to resolve an infection by a recombinant vaccinia virus expressing OVA protein.

Molecular analysis of the CYP1B1 gene: identification of novel truncating mutations in patients with primary congenital glaucoma.

BACKGROUND: Mutations and polymorphisms have been identified in the CYP1B1 gene; while mutations that affect the conserved core structures of cytochrome P4501B1 result in primary congenital glaucoma (PCG), mutations in other regions hold the potential to define differences in estrogen metabolism. In the present study, we analyzed the CYP1B1 gene in Mexican patients with PCG and described four novel mutations. MATERIALS AND METHODS: The sample included 12 nonrelated cases with PCG. Analysis of coding regions of the CYP1B1 gene was performed through PCR and DNA sequencing analysis from genomic DNA. RESULTS AND DISCUSSION: Molecular analysis of the CYP1B1 gene showed the following molecular defects: (1) a novel single-base pair deletion within codon 370 (1454delC) that produces a substitution of leucine instead of proline and a premature stop codon 57 amino acids after the last original amino acid; this family also harbored a novel polymorphic variant of the cytochrome P4501B1 with six single-nucleotide polymorphisms (142C-->G; 355G-->T; 729G-->C; 4326C-->G; 4360C-->G and 4379C-->T); (2) a novel single-base pair deletion within codon 277 (1176delT) that results in a premature stop codon; (3) a novel single-base pair deletion within codon 179 (880delG) that produces a substitution of arginine instead of alanine and a premature stop codon 17 amino acids downstream from the last original amino acid, and (4) a duplication (or insertion) of ten base pairs within codon 404 (1556dupATGCCACCAC) that results in a premature stop codon 26 amino acids after the last original amino acid. We also observed in 2 nonrelated patients a deletion of 13 bp (1410_1422delGAGTGCAGGCAGA) previously reported for other populations. CONCLUSION: We reported four novel mutations and a novel polymorphic variant in the CYP1B1 gene in PCG in the Mexican population; it has important implications in diagnosis and genetic counseling.

Haematological response to haem iron or ferrous sulphate mixed with refried black beans in moderately anaemic Guatemalan pre-school children.

OBJECTIVE: Combating iron deficiency in toddlers with iron-fortified food has proved difficult in countries with phytate-rich diets. For this purpose, a new haem iron preparation was developed. The study compared changes in iron status after administration of refried beans with beans fortified with a haem iron preparation or ferrous sulphate (FeSO4). DESIGN: In a masked, stratified-randomised intervention trial, children received five 156-g cans of refried black beans per week for 10 consecutive weeks. The beans-only (control), FeSO4 and haem iron groups were offered a cumulative dose of 155 mg, 1625 mg and 1700 mg of iron from the bean intervention, respectively. Haemoglobin (Hb) and ferritin concentrations were determined at baseline and after 5 and 10 weeks. Compliance was examined weekly. SETTING: A low-income community in Guatemala City. SUBJECTS: One hundred and ten children aged 12-36 months with initial Hb values between 100 and 115 g l(-1). RESULTS: The cumulative intake of beans was approximately 80% of that offered, signifying an additional approximately 1300 mg of either haem or inorganic iron in the corresponding treatment groups over 10 weeks. Hb concentrations increased by the order of 7.3-11.4 g l(-1) during the intervention, but without significant differences across treatments. Average ferritin concentrations were unaffected by treatment assignment. However, post hoc analysis by subgroups of initial high ferritin and initial low ferritin found the Hb increments after 10 weeks in the haem iron group (13.1+/-7.7 g l(-1)) to be significantly greater than the respective increases (6.8+/-11.2 and 6.4+/-8.5 g l(-1)) in the inorganic iron and beans-only groups. CONCLUSIONS: Canned refried beans are a candidate vehicle for fortificant iron. Given the improved colour and organoleptic properties imparted by haem iron added to refried beans, its additional potential for benefiting the iron status of consumers with iron deficiency may recommend it over FeSO4.

Caracterización de recursos físicos y humanos en diversos servicios de rehabilitación / Characterization of physical and human resources in different rehabilitation services

El presente estudio responde a una necesidad a nivel nacional y a un interés de las investigadoras de contar con datos respecto de los recursos existentes en el área de la Medicina Física y Rehabilitación del Sistema Público de Salud. Es por esto, que en conjunto con la Unidad de Rehabilitación del Ministerio de Salud, se dirigieron los esfuerzos hacia la creación de un estudio de tipo descriptivo y transversal, que tuvo el objetivo de caracterizar los recursos físicos, de equipamiento y humanos con que cuenta el Universo de los Servicios de Medicina Física y Rehabilitación y las Unidades de Kinesiología de los Hospitales Públicos, Centros de Diagnóstico terapéutico (CDT) y Centros de Referencia de Salud (CRS) de la Región Metropolitana, que corresponden a 20 Establecimientos. Para la medición se utilizó un cuestionario autoadministrado, el cual fue construido basándose en la Guía Metodológica de Estudio de la Red Asistencial, elaborada en el año 1997 por el DINRED del Ministerio de Salud. Posteriormente se realizó un pretest, luego del cual fue necesario realizar una segunda adaptación de dicho instrumento, obteniendo así el cuestionario que fue aplicado al universo de los Jefes y Funcionarios de dichos Servicios y Unidades del Subsistema Público, en la Región Metropolitana. Este instrumento midió la dotación de los recursos antes mencionados y además las percepciones que tenían los encuestados sobre el Servicio o Unidad donde desempeñan su trabajo, esto tuvo como propósito identificar algunos aspectos culturales y motivacionales del recurso humano que pueden afectar el desempeño global de su lugar del trabajo. Como conclusión, se logró realizar una caracterización de los recursos físicos, de equipamiento y humanos, según los jefes y funcionarios. Además se logró caracterizar la percepción de los funcionarios y jefatura con respecto a tres dimensiones del Servicio o Unidad: Desarrollo Organizacional, Recursos Humanos, Recursos Financieros y de Equipamiento. Adicionalmente sólo las jefaturas describieron aspectos estructurales de su Servicio o Unidad como por ejemplo: tipo de establecimiento, estado de la estructura, instalaciones, entre otros.

Impaired temporo-occipital blood flow in an atypical CLN1 case with late infantile onset and granular osmiophilic deposits.

A 5-year-old boy presented with frequent absences. Speech began to regress. He became ataxic, barely able to walk. Studies with Xe-133 and hexamethylpropylene amine oxime single-photon emission computed tomography revealed sharply decreased cerebral blood flow, especially in the occipital area. Landau-Kleffner syndrome was suspected but a sleep electroencephalogram showed few abnormalities. He was started on clorazepate and diltiazem. A skin biopsy to rule out possible CLN2 revealed, instead of the predicted curvilinear profiles, granular osmiophilic deposits, consistent with infantile neuronal ceroid lipofuscinosis (CLN1). The family reported increased seizure frequency and consulted with a colleague, who advised them to resume valproate and discontinue diltiazem. The boy died shortly thereafter. Decreased cerebral blood flow is a new finding in CLN1 with delayed onset. Calcium-channel blockers improve cerebral blood flow and perhaps delay clinical regression.

Neuroanatomy of the self: evidence from patients with frontotemporal dementia.

OBJECTIVE: To evaluate the frequency and types of change in "self" seen in frontotemporal dementia (FTD) and to determine the relative involvement of the nondominant and dominant frontal and temporal brain regions in FTD patients with or without changes in a sense of self using neuropsychology tests and neuroimaging. BACKGROUND: The self has been defined as "the total, essential, or particular being of a person" involving "the essential qualities distinguishing one person from another." Some suggest that the frontal lobes play a dominant role in maintaining the self. FTD affects anterior frontal and temporal areas and can be associated with a loss of self. METHODS: Seventy-two consecutive FTD patients were evaluated with neuropsychiatric, neuropsychologic, and behavioral measures. Patients were imaged with MRI and SPECT. Charts were reviewed by a social psychologist to determine patients who exhibited a dramatic change in their self as defined by changes in political, social, or religious values. The brain areas with the most severe atrophy or hypoperfusion on neuroimaging were noted. RESULTS: Seven of 72 patients exhibited a dramatic change in self. In six of the seven, the selective dysfunction involved the nondominant frontal region. In contrast, only one of the other 65 patients without selective nondominant frontal dysfunction showed a change in self. CONCLUSIONS: FTD patients with asymmetric loss of function in the nondominant frontal lobe often exhibit a diminished maintenance of previously learned self-concepts despite intact memory and language. Normal nondominant frontal function is important for the maintenance of the self.

Sentinel lymph node mapping in melanoma with technetium-99m dextran.

PURPOSE: The aim of this work is to evaluate the capability of Tc99m B Dextran as a lymphoscintigraphic agent in the detection of the sentinel node in skin lesions. MATERIALS AND METHODS: Forty-one patients with melanomas (39) and Merkel cell tumors (2) had perilesional intradermal injection of Tc99m-Dextran 2 hours before surgery. Serial gamma camera images and a handheld gamma probe were used to direct sentinel node biopsy. RESULTS: In 39/41 patients, lymph channels and 52 sentinel nodes (one to three sentinel nodes/patient) could be visualized. In one patient, with a dorsal melanoma, no lymph channels or lymph nodes could be demonstrated on the images and only minimal radioactivity was found in the regional nodes with the probe. Another patient with a facial lesion failed to demonstrate lymph channels or nodes. No adverse reactions were observed. CONCLUSION: Tc99m-Dextran provided good definition of lymph channels and sentinel node localization, without the risks related to the use of potentially hazardous labeled materials of biological origin.

Using recombinant coxsackievirus B3 to evaluate the induction and protective efficacy of CD8+ T cells during picornavirus infection.

Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were approximately 1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in approximately 50-fold-lower virus titers in the heart and approximately 6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.