SPMicros was born in the year 1966 and since then the Society has promoted 46 annual Congresses. These have been long recognized as a nursery for young scientists with a special interest in microscopy. The meetings of the Society, fostered by high level scientists like David Ferreira and Teixeira da Silva, to mention only the ones that have left us this year, were once marked by a exigent standard of quality that left a lasting mark in all of us.

A variety of frequently encountered clinical problems lend themselves readily to investigation by analytical electron microscopy. e.g., a combination of scanning or transmission electron microscopy and energy dispersive x-ray microanalysis. The most common application is identification of xenobiotics or exogenous substances, such as localization and quantitation of inorganic particulates in lung tissues in patients with pneumoconiosis; identification of foreign materials within granulomas; and analysis of foreign bodies. Electron probe X-ray microanalysis (EPXMA) is also useful in the study of tissue reactions to various surgical implants of foreign materials. A variety of metals and other elements may be detected with energy dispersive X-ray analysis, including copper in tissues of patients with Wilson’s disease, thorium and gadolinium in patients injected with radiographic contrast agents (Figure 1), or gold in patients treated with long-term chrysotherapy. Endogenous particulates such as urinary calculi (Figure 2), gallstones, intraarticular and periarticular crystalline deposits in patients with rheumatic disease, dystrophic or metastatic calcifications, and hemosiderin may be analyzed rapidly and efficiently by means of EDX. Certain organometallic drugs such as amiodarone (iodine) or sodium stibogluconate (antimony) may also be detected in human tissues. Analytical electron microscopy has been a useful adjunct to forensic pathology for many years in diverse areas such as identification of trace evidence constituents or detection of arsenic or lead in victims with heavy metal poisoning. The detailed elucidation of anatomic, physiologic, and pathologic conditions provided by analytical electron microscopy is a useful diagnostic and investigative tool in clinical medicine; the analytical results often have diagnostic, therapeutic, and/or medicolegal implications. This imaging technology should grow in utility in the future as it is complemented by other techniques such as mass spectrometry, and laser Raman and infrared microspectroscopy.

In this talk the utility of atomic force microscopy (AFM) for research into infectious parasites will be discussed. AFM has grown from relatively recent beginnings to become an extremely powerful technique in the life sciences, coupling high resolution imaging with a range of non-imaging experiments. Importantly, these experiments can be performed in situ, even on individual molecules or on live cells.

The two examples discussed relate to the important diseases leishmaniasis and malaria. Leishmaniasis is a disease caused by the protozoan parasite of the Leishmania genera, and causes approximately 60,000 deaths per year. Despite the high death toll, the disease has been the subject of relatively little research and little treatment is available, probably because the most severe cases are confined to developing nations. The most severe form, visceral leishmaniasis is caused by the species known as Leishmania infantum (syn. L. chagasi). A promising new anti-leishmania drug, DS01 has been recently isolated from amphibian secretions and can kill L. infantum in low concentrations. We were able to culture and prepare for microscopy L. infantum promastigotes for the first time, as well as to study the effects of DS01 on cell morphology and membrane integrity. The results from both AFM and SEM are highly complementary and illustrate the possibility of membrane-focussed activity as well as the possibility of attack on the flagella (figure 1).

Malaria is one of the most deadly diseases in the world, killing more than 600,000 people per year, mostly in low-income countries. It is caused by Plasmodium parasites, and the most commonly studied stage is that in which the parasite invades the blood. Prior to blood invasion, the parasites infect hepatocytes in the liver, with formation of a parasitophorous vacuole, where they develop into exoerythrocytic forms and multiply to generate thousands of merozoites, later released into the bloodstream and causing disease. However, infection of liver cells, which is clinically silent, is required for disease progression. We studied infection of liver cells by Plasmodium using combined epifluorescence and atomic force microscopy. We observed significant changes in cell morphology as infection progressed (figure 2). Furthermore we made nanoindentation measurements with the AFM, to determine cellular stiffness. We observed stiffening of the cells after 48 hours of infection compared to uninfected cells. This was a cellular response to the Plasmodium infection, rather than a result of the stiffness of the invading parasites themselves. This stiffening may be caused by reinforcement of cytoskeletal structures, and we believe this may reflect a self-defence mechanism by the cell itself.

The coccolithophores are a marine phytoplankton group that can play an important role in selective uptake of Cadmium (Cd) in neritic environments. Under project Cd-ToxCoN (PTDC/MAR/102800/2008) studies were conducted to investigate the in vitro reaction of Emiliania huxleyi to Cd exposure, namely changes in the crystal lattice of (cocco)liths, the calcite elements of the exoskeleton (coccosphere).

Uni-algal cultures of E. huxleyi were obtained by multiple cell isolation from the same water sample collected from Portuguese offshore waters, during opportunity cruises (Portuguese Marine Navy). Several morphotypes of E. huxleyi are currently recognized. The identification of the morphotype of E. huxleyi from Portuguese coastal waters was done by morphological analysis of the exoskeleton using scanning electron microcopy (JEOL JSM-5200LV and FEG-SEM JEOL 7001F) and by DNA sequencing of tufA gene following.

Experiments were conducted in batch cultures grown in enriched sea-water medium (K/10) under constant environmental conditions (14h L: 10h D, 15ºC, 40 µmol photons m-2 s-1). At the exponential growth phase, 3 replicate E. huxleyi cultures were subject for 48h to Cd concentrations of 10 µgL-1 and 100 µgL-1, two and three orders of magnitude above average marine concentration values respectively. In each case three additional replicate cultures with no Cd added were used as control.

The effect of Cd was evaluated by measuring in vivo fluorescence (ratio of variable (Fv) to maximum (Fm) fluorescence) (Water PAM fluorometer – Walz) and by detailed morphometric analysis of coccospheres and liths performed on SEM micrographs. The parameters measured are those presented in Figure 1. Malformed, damaged or tilted liths were not measured.

Morphological observations of the coccosphere and liths and DNA sequencing allowed the identification of the isolated strains of E. huxleyi as morphotype A. Results on the Cd exposure experiments indicate that E. huxleyi type A seems to tolerate high concentrations of Cd. Despite the very high concentration of Cd tested no lethal limit was reached and Fv/Fm values recorded after 48h at 10 µgL-1 Cd (0.607±0.008) and 100 µgL-1 Cd (0.603±0.008), very similar to the values recorded in the control cultures (0.642±0.016 and 0.636±0.018, respectively) and before Cd addition (0.642±0.012 and 0.636±0.007, respectively).

Regarding the different morphometric parameters analysed on coccospheres and liths, no significant measurable effects were observed. However, we observed, in response to increasing Cd concentration, an increasing number of liths with fused or partially fused elements (Fig. 2) suggesting a higher calcification of liths. This interpretation is supported by the results of the analysis of coccosphere calcium content. Cultures exposed to Cd presented a higher Ca content compared to control cultures. The highest values were recorded in coccospheres submitted to 100 µg.L-1 of Cd representing a 65% increase in Ca content in comparison to control coccospheres.

E. huxleyi is ubiquitous in present day oceans and usually very tolerant to culture conditions and is thus frequently used as a model species in the study of coccolithophores. These same characteristics may also justify the observed high tolerance to Cd. Studies with other species are needed to clarify if the surprising resistance to Cd is unique to E. huxleyi or characteristic of other unicellular algae with calcium carbonate shells.

This work was funded by projects PEst-OE/Mar/UI0199/2011, Pest/OE/CTE/UI/0263 and PTDC/MAR/102800/2008

2

A long time objective of ophthalmologists is to diagnose corneal cells dysfunction prior to its pathologic expression. With this motivation, we are currently developing a new instrument for in vivo metabolic imaging of corneal tissues.

Metabolic alterations are known to be the first sign of several corneal pathologies and can be assessed through non-invasive monitoring of metabolic co-factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The quantification of the relative proportions between free and protein-bound NADH and FAD can be achieved using fluorescence lifetime-resolved methods. This approach has already been applied in age-related macular degeneration, diabetic retinopathy and epithelial cancer.

FAD and NADH imaging can be performed by one-photon excitation (1PE) and two-photon excitation (2PE) techniques. The latest has the advantage of allowing simultaneous excitation of both metabolic co-factors. However, there are still safety concerns when considering in vivo ocular studies in humans using 2PE.

Due to these concerns we used, as a first approach, a 1PE system for evaluating the feasibility of corneal FAD imaging. The use of FAD has advantages over NADH. It can be excited over longer excitation wavelengths, is more resistant to photo-bleaching and is located exclusively in the mitochondrial space.

A PicoQuant MicroTime 100 (PicoQuant GmbH, Berlin, Germany) coupled to an Olympus BX51 Microscope (Olympus Corporation, Tokyo, Japan) was used to monitor FAD autofluorescence. The instrument uses a 440 nm pulsed diode laser (330 ps) running at a pulse rate of 40 MHz. The instrument was modified by us to allow the acquisition of both fluorescence lifetime and reflectance images and to enhance scattered light rejection.

Intensity decay curves were processed with SymPhoTime v5.3 Software (PicoQuant GmbH, Berlin, Germany). The fluorescence decay times were obtained after applying a non-linear least square fit to the decay data and the goodness of fit was evaluated by the analysis of the residuals and the chi-squared (χ2).

We have acquired fluorescence lifetime images of ex vivo healthy Wistar rat corneas (Fig.1) using two different instrument setups: 1- using the emission filters provided by the manufacturer; 2- placing extra emission filters to fully reject the scattered excitation light. In both setups, FAD fluorescence data presented a bi-exponential decay with a short (protein-bound FAD) and a longer (free FAD) lifetime component.

While both setups provide FAD fluorescence decays, only the second retrieves valid metabolic information. We obtained two lifetime components, one of 0.118 (0.028) ns and another of 2.11 (0.16) ns, with a relative contributions of 39.4 (2.2) and 60.6 (2.2), respectively. These values are in accordance with the literature.

Corneal layer discrimination is possible based on morphologic characteristics. However, the fluorescence lifetime images do not provide morphological detail (Fig.1), possibly because FAD is only present in the mitochondria. These organelles are small and tend to accumulate around the nuclei.

So, we modified the instrument’s optical setup to allow the acquisition of both fluorescence lifetime images and reflectance images. Figure 2 shows an example of the corneal epithelial layer.

The image resolution and depth penetration are still not ideal. Since the assessment of corneal endothelial layer metabolic function is also within our goals, we are currently implementing further modifications to improve both the instrument’s resolution and depth penetration.

The characterization of FAD fluorescence lifetime in unhealthy corneas is important to detect corneal dysfunctions prior to its pathologic expression. Therefore, we intend to study metabolic altered Wistar rat corneas. The alterations will be induced by potassium cyanide, which is a reversible inhibitor of the fourth complex of the mitochondrial electron transport chain.

Financial support received from the Fundação para a Ciência e a Tecnologia under the research projects PTDC/SAU-BEB/104183/2008 and PTDC/SAU-ENB/122128/2010.

The therapeutic usefulness of anticancer agents relies on their ability to kill cancer cells while sparing normal cells and tissues. Ruthenium complexes hold great potential as suitable alternative drugs to the current cisplatin in the treatment of cancer. Our approach in this field has been the study of organoruthenium complexes, [RuII(η5-Cp)] family, namely [RuII(η5-Cp)(bipy)(PPh3)]+ (PPh3 = triphenylphosphine; bipy = 2,2’-bipyridine), TM34. This compound was tested for antitumor activity against tumor cells representative of human cancer diseases. A large spectrum of activity was found, largely surpassing cisplatin in efficacy (Fig 1).

How a drug distributes and localizes within cells is of fundamental importance as the drug must concentrate in the compartment that houses its target(s). The importance of cellular enzymes on the effects of anticancer drugs for which multiple biological pathways have been proposed led us to address the involvement of the lysosomes and lysosomal enzymes (AcPases) in the mechanism of cell death.

The cytotoxicity of TM34 was tested using the MTT assay. TM34 cellular distribution was measured by ICP-MS. The effect of TM34 on the activity of acid phosphatase (AcPase) was evaluated by the hydrolysis of p-nitrophenyl phosphate to p-nitrophenol. AcPase localization was investigated by TEM using the Record and Griffing (1988) cerium-based method.

Results showed that the sensitivity of AcPase assay is higher in comparison to cell proliferation assays based on the reduction of tetrazolium salts (MTT) (Fig 2). From TEM images AcPase reaction product appears as dark electron dense deposits in the lysosomes and dictiosomes. After 3h treatment TM34 provokes disruption and vesiculation of the Golgi apparatus, although some reaction product is present in the lysosomes (Fig 2).

The lysosome could be considered a possible target for TM34. Morphological evidence was observed for the participation of the Golgi apparatus in the vesiculation induced by the compound.

Supported by the Fundação para a Ciência e Tecnologia (FCT): PTDC/QUI-QUI/101187/2008.

Tuberculosis (TB) is one of the major causes of mortality and morbidity worldwide accounting for 3.1 million deaths per year. This disease, caused by Mycobacterium tuberculosis (M. tuberculosis) made a deadly comeback, during the 1990’s, triggered mainly by the emergence of acquired immunodeficiency syndrome (AIDS). More recently, the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) M. tuberculosis strains, uncovered the most freighting face of this disease an incurable infection with the currently available therapeutic tools. Although Portugal is considered a medium incidence setting, annually are reported MDR and even XDR TB cases. The majority of these cases occur in the Lisbon area and the strains involved are genetically related being known as Lisboa family.

In the present work a group of 283 M. tuberculosis isolates collected in a Lisbon hospital during a two years period (2008-2009) were studied. The morphology of colonies grown on Lowenstein-Jensen slants was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) using previously described procedures. The aim of the study was the establishment of a link between mycobacteria drug susceptibility and structure. In the first part of the study approximately 20 isolates, with different drug susceptibility profiles ranging from pan-susceptible to XDR, were grown on Lowenstein-Jensen slants and their morphology was compared. Although all mycobacteria originated rough colonies their size differ with the drug susceptibility profile. The pan-susceptible strains generated larger colonies than drug resistant strains as shown in figure 1.These colonies were then processed for SEM analysis. The results obtained show that mycobacteria surface are distinct in susceptible and drug resistant strains as shown in figure 2.A and B. While drug susceptible mycobacteria have a homogenous surface (Figure 1A), drug resistant bacteria present a heterogeneous surface (Figure 2B) with small protrusions (Fig. 2B inset). In order to evaluate the existence of differences in the ultrastructure of circulating M. tuberculosis strains the colonies were processed and analysed by TEM. For this approach were selected only two isolates: the pan-susceptible R188/09 and the XDR HPV108/09.

The results obtained by the analysis of at least 300 bacteria present in non consecutive sections show that mycobacteria cell width (0 350 nm) is similar for both bacteria (Table 1). Nevertheless, their cell length and cell envelope width are significantly different. The XDR strain is shorter (p=0.009) and has a ticker cell envelope (p=0.004) than the pan-susceptible strain. These results are in agreement with those published in the literature.

Altogether our data clearly shows the existence of a link between mycobacteria ultrastructure and drug susceptibility. In order to better evaluate these differences a larger number of isolates must be studied. The use of other electron microscopy techniques, such as CEMOVIS, will avoid the formation of undesirable artefacts (e.g. mesosome) produced by dehydration and room temperature sectioning allowing a better characterization of mycobacteria ultrastructure.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia (SFRH/BD/73579/2010, C2008-C2008_P2 and PEst-OE/CTM-UI0084/2011 grants.)

African Swine Fever Virus (ASFV) is one of the most threatening agents of domestic pig diseases, without a vaccine or treatment, being its control exclusively based on compulsive sanitary measures. Until now, ASFV was thought to perform its viral life cycle within the cytoplasm although recent evidences indicate the presence of viral DNA material inside the host cell nucleus promoting ASFV reclassification into the Nucleocytoplasmic Large DNA Viruses group (NCLDV). So far, no studies have been performed regarding ASFV genome replication phenomena or the host nuclear compartments morphology during cellular infection. Therefore, we aim to unveil the spatiotemporal localization of ASFV DNA replication and the distribution patterns of three subnuclear domains (PML, speckles and coiled bodies), in order to improve knowledge on host nucleus-viral interactions.

For the viral DNA replication foci staining, Vero cells were synchronized at a G2/M stage with nocodazole (250 ng/ml, 24h). Following synchronization, cells were grown on glass coverslips (5,0x104 cells/cm2) and infected with ASFV-Ba71V isolate (1h adsorption period with a multiplicity of infection of 5). Afterwards, at specific time-points of viral infection, BrdU (150 µM/ml) was added for a short pulse (30 min) and immediate fixation was performed. For BrdU and ASFV immunodetection, the following primary and secondary antibodies were used: sheep polyclonal anti-BrdU (GTX21893, Genetex, USA; 1:100), an in-house clarified swine anti-ASFV whole-serum (1:100); Alexa Fluor 594 donkey anti-sheep IgG (A-11016, Life Technologies, USA; 1:500) and a FITC rabbit anti-swine IgG (ab6773, Abcam, UK; 1:400). For PML, speckles and coiled nuclear bodies immunolabeling a rabbit polyclonal anti-PML (ab53773, Abcam; 1:100), a goat polyclonal anti-SC35 (sc-10252, Santa Cruz Biotech, USA; 1:50) and a rabbit popyclonal anti-coilin (sc-32860, Santa Cruz Biotech; 1:50) were used, while DyLight 594 donkey anti-rabbit IgG (ab98490, Abcam; 1:500) and Alexa Fluor 594 chicken anti-goat IgG (A-21468, Molecular Probes, 1:400) were used as the secondary antibodies. Microscopic analysis of cells was performed with a Leica epifluorescence microscope (model DM R HC, Germany). De novo synthesized viral DNA was identified in a scattered nuclear localization (discreet dots) during the initial period of infection (Fig. 1, row 1), whereas in a later phase of infection (8h pi), most of the BrdU signal accumulated in the cytoplasmic viral factory (Fig. 1, row 2). PML bodies and nuclear speckles presented a morphological enlargement and a decreasing number in ASFV infected Vero cells (Fig. 2, rows 1 and 2). In an opposite manner, the coiled bodies increased their number during infection (Fig.2, row 3).

Our results provide the first evidence that ASFV DNA replication also occurs inside the host nucleus. Nuclear discrete replication foci, at the initial onset of the viral infection, contrast to an accumulation of synthesized viral DNA inside the bigger cytoplasmic viral factory, suggesting that these two distinct patterns are related to the viral life cycle demands. The early PML reorganization can probably be related with cellular antiviral defense mechanisms, given that DNA damage response and p53 are activated during ASFV infection. The low number of nuclear speckles observed in ASFV-infected cells, associated with their enlargement, is most possibly related to the relocation of host splicing factors, since ASFV genome lacks intronic regions.

This study was supported by Fundação para a Ciência e Tecnologia through the Project (PTDC/CVT/105630/2008) and the PhD fellowship (SFRH/BD/65532/2009).

Animal skin pigments, often of undisclosed biochemical nature, have many functions besides signalling and camouflage: they also play a protective role against environmental stressors, from radiation and oxidative stress–inducers to predators and parasites. Most pigments are not fully synthetized by animals but rather acquired from vegetable feed and often biotransformed. These intriguing pigments called the attention of researchers in the past, such as the awkward green “chlorophylloid” pigments of the marine invertebrate Bonellia, first described in the 19th century. For decades, however, research has come to a stand–still. The marine annelid Eulalia viridis (Phyllodocidae: Polychaeta), although common throughout NW Europe, has so far received little attention to the origin and function of its bright green colouration. In fact, with the exception of part of its digestive system even the basic histological description of this animal is absent, as for marine annelids in general.

Adult wild E. viridis were subjected to a comprehensive microscopy study employing several fixation and staining techniques for bright–field (BF), transmission electron (TEM) and UV–epifluorescence microscopy in order to disclose the pigment cells’ microanatomy and arrangement and to infer the potential origin of the pigments. Stains for bright–field microscopy included Haematoxylin and Eosin (HE), Alcian Blue–Periodic Acid/Schiff’s–Haematoxylin (AB–PAS–H) and Bronner’s Sudan Black. Samples were either embedded in paraffin (BF and UV microscopy) or LR White resin (BF and TEM).

The high complexity of the animals’ integument was confirmed, in accordance to the scarcely known polychaete microanatomy. The green colouration is chiefly caused by the presence of specialized skin cells containing minute pigment vesicles (Fig. 1A). These pigment cells are compressed between multiple types of skin cells and possess microvilli that protrude through the integument’s polysaccharide–based cuticle. The vesicles themselves appear attached to the cells’ tonofilaments (Fig. 1B). The vesicles are densely packed within pigment cells (Fig. 2A) and are not fluorescent, which should exclude the existence of active chlorophyll within (Fig. 2B). From this data is may be inferred that the pigments are chlorophyll–derived substances (chlorins/pheophorbides), similar to what was observed in the polychaete Chaetopterus variopedatus. Oddly enough, while the latter is a filter–feeder, thus able to sequester microalgae from the water column, E. viridis is a foraging scavenger that feeds mostly on the flesh of other invertebrates. Furthermore, the animal inhabits nude rocky shores and not algal beds where its green colour would be a clear advantage, which renders further intriguing the adaptative value of this feature, however, due to some known biocidal properties of these pigments; defence should not be overruled.

P.M. Costa acknowledges the Portuguese Science and Technology Foundation (FCT) for the grant SFRH/BPD/72564/2010. The authors also thank L. Ascensão (FCUL) for the important assistance.

Marine fungi occur either in Open Ocean or in the intertidal zone of sandy beaches, salt marshes and mangroves, where their hosts and substrates are found. The development of morphological adaptations like appendages and sheaths of the spores are vital to the settlement and attachment to substrate surfaces, floatation and dispersion on seawater. The morphological features of these appendages and sheaths of spores also have an important role in the identification of marine fungi. Differential interference contrast (DIC) microscopy is an essential tool for the observation of these mucilaginous structures in marine fungi spores and was therefore applied to marine mycota from surveys along the Portuguese coast.

Since 1991 Portuguese marine fungi have been studied and characterized based on morphological identifications. The studied environments included salt marshes, sandy beaches and marinas. On these environments different substrates were collected such as plants and baits of Spartina maritima, different categories of drift substrates and Fagus sylvatica and Pinus pinaster baits. These substrates, which had been exposed to different conditions of permanent and temporary submersion, were subjected to an initial examination under the stereoscope microscope in order to detect fruit bodies and spores of marine fungi. These structures were then observed in order to achieve the taxonomic identifications of these fungi, based on dichotomous keys for marine fungi.

To observe and characterize the wide variety of appendages and mucilaginous sheaths of the ascospores, basidiospores and conidia often invisible in the bright field of the light microscope, the use of DIC microscopy was implemented, because three-dimensional images are produced, highlighting them. The identified fungi were microphotographed with a Leica Wild MPS 52 with Fujichrome RTP- 135, 64T Tungsten.

The studies carried out with substrates subjected to conditions of permanent submersion highlighted the dominance of Ascomycota with unitunicate asci. The unitunicate asci are thin - walled, persistent or early deliquescing, favoring ascospores release on marine environmental conditions (fig 1a, 1b and 1c). On the other hand, Ascomycota with bitunicate asci, were mainly detected on temporary submersion conditions. These fungi presented asci with active spore discharge and mucilaginous sheaths that contributed to substrate attachment (fig. 2a and 2b). Ascospores and conidia presented morphological diversity on their appendages, which has particular importance on their success in marine environment (figs. 3, 4, 5, and 6).

Marine Mycologists are in agreement that the best technique to observe the appendages and sheaths of spores, often invisible in the bright-field microscope is the use of differential interference or phase contrast microscopy. DIC microscopy was then applied to the observation of Portuguese marine fungi enabling to thoroughly characterize the structures essential to their morphological identifications.

The identification of genome-wide expression profiles that discriminate different molecular subtypes of diseases became a recent field of research, with a visible impact in cancer research. In breast cancer, molecular immunophenotyping of the tumor is critical to support therapeutic decisions.

Over the last years, some studies revealed that a high percentage of Feline Mammary Tumours (FMTs) display fHER2 overexpression followed by a very short survival period, like in woman HER2-positive breast cancer. Usually, FMTs are malignant and very aggressive (85% to 93%), showing bad prognosis. In feline oncology, the histopathological classification is the only possible option, meaning that little data about molecular expression profiles is available. Given these facts, our study aims to confirm if primary tumor immunophenotype is conserved at regional and distant metastasis.

Two primary mammary carcinomas, their regional and distant metastasis were collected during necropsy examination of two queens. The immunohistochemical analysis measured HER2 status, progesterone receptor (PR), estrogen receptor (ER), cytokeratin (CK) expression levels and the Ki67 index in all samples using the antibodies: anti-HER2 (A0485, DAKO); anti-PR (1E2, Ventana); anti-ER (SP1, Ventana); anti-CK (AE1/AE3, DAKO) and anti-Ki67 (MM1, Leica). The fixation and the interpretation criteria followed the American Society of Clinical Oncology guidelines.

The first tumor was an anaplastic mammary carcinoma with high malignancy grade (III) both in the primary tumor and in metastatic lesions (Fig.1). Immunophenotyping revealed HER2 and PR positivity in both primary mass and regional metastasis (Fig.1 - A4, B4; A5, B5), but ER negative staining in these locations (Fig.1- A6, B6). Distant metastasis showed a lack of PR expression (Fig.1 - C5), but maintained equal status of the two other markers (Fig.1 - C4, C6). Similar Ki-67 index values were determined in primary tumor (0.26) and in metastasis (0.19). The second FMT was a tubulopapillary carcinoma (Fig.2) with high malignancy grade (III) and, unlike, the first tumor, immunolabeling for PR was positive both in the primary tumor and in the different metastasis, revealing molecular pattern conservation. The Ki-67 index was always high either in primary tumor (0.33) as in regional (0.48) and distant metastases (0.41) (Fig.2 - A3, B3, C3). All tissue samples were positive for CK, confirming their epithelial nature (Fig.1 - A2, B2, C2 and Fig.2 - A2, B2, C2).

Our study points out that FMTs molecular expression patterns may not be preserved in primary tumor and metastatic lesions. If we just looked to the immunophenotype of the primary mass and the regional metastasis, both carcinomas would be classified as luminal B subtype (ER-, PR+ and HER2+). However, when we evaluated the distant metastasis, while the immunophenotype of the second FMT was conserved, the PR expression in the first FMT was lost, which implied a tumor reclassification to HER2 subtype. In women, both subtypes have anthracycline-based regimens prescription but luminal B subtype also benefits with hormonal therapy, while the HER-2 subtype has indication for specific immunotherapy. Similarly to women breast cancer, the molecular classification of feline mammary tumors should be performed to improve treatment-response and animal live quality.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through PhD fellowship SFRH/BD/70720/2010.

Helicobacter pylori is a pathogenic spiral-shaped, microaerophilic, gram-negative bacterium, that inhabits the human stomach. Infection is usually acquired during childhood and always elicits an acute immune response that is, however, inefficient in bacteria clearance. Therefore, in the absence of effective treatment, infection and gastritis (non ulcer dyspepsia, NUD) persist throughout the patient’s life. Depending on its severity and pattern, in about 15% of infected adults, this silent destruction of the gastric mucosa may further progress to peptic ulcer disease (PUD) (gastric and duodenal ulcers, GU and DU respectively) and/or gastric cancer. Infection with H. pylori is also the major cause for the development of paediatric PUD, a rare event that may occur shortly after infection. In addition to the still undisclosed genetic susceptibility of these young patients, the virulence of the implicated H. pylori strain plays a crucial role in the paediatric PUD pathogenesis. Recently, we proved by in vitro infection assays that, compared with paediatric NUD-associated isolates, a group of paediatric ulcerogenic-strains present a greater ability to induce a marked decrease in the gastric cells viability and to cause them severe cytoskeleton damage and mucins’ production/secretion impairment. Moreover, we showed that their enhanced virulence result from a synergy between the ability to better adapt to the hostility of their niche and the expression of cagA, vacAs1, oipA ‘‘on’’ status, homB and jhp562 virulence factors. Accordingly, these ulcerogenic strains share a particular proteome profile, providing them with better antioxidant defences, a metabolism favouring the biosynthesis of aromatic amino acids and higher motility.

We are now characterizing/comparing the cellular and flagella morphologies of H. pylori strains isolated from Portuguese children, associated with DU, GU or NUD, belonging to the vast and multiethnic collection of the Instituto Nacional de Saúde Dr. Ricardo Jorge (Portugal). For that, bacteria were grown in H. pylori selective medium (Biogerm, Maia, Portugal) at 37ºC in a microaerobic environment (Anoxomat®, MART Microbiology BV, Drachten, The Netherlands) for 24 h. For Leifson staining analysis, a drop of each bacterial suspension (in PBS) was spread in cleaned microscope slides, stained with the Leifson dye solution until a golden film developed on the dye surface and a precipitate appeared throughout the sample, and analysed by optical microscopy. For Transmission-Electronic-Microscopy (TEM) studies bacterial pellets were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.

Corroborating the better swimming abilities of the PUD strains, as previously shown by motility assays, optical microscopy analysis of Leifson stained slides demonstrated marked differences in the morphology of the studied strains (Figure 1). The H. pylori strain associated with DU (Hp 1152/04) seem longer than all the others and, in contrast, that associated with GU (Hp 499/02) is the shortest one and presents a, more pronounced, spiral morphology. Moreover, our preliminary data on TEM analysis indicate the presence of more abundant and apparently more organized flagella in the GU-associated strain Hp 499/02, in contrast to the NUD control strain, Hp 655/99 (Figure 2).

Work supported by Research Grant 2011 – Sociedade Portuguesa de Gastrenterologia.

The oral cavity is susceptible to several calcifications such as salivary calculi (sialoliths), dental calculus (tartar) and tonsillar concretions (tonsilloliths). Although several individual studies had been already carried out, a comprehensive morphological and elemental comparison between them is still missing.

Sialoliths are most commonly found in the submandibular glands and are composed of regions rich in Ca and P minerals, namely hydroxyapatite, whitlockite and brushite, and regions consisting of organic matter with high-sulphur content. These regions are organized in alternating concentric layers. Several bacterial species have also been identified in sialoliths microstructure showing that infection occurs recurrently throughout the stone formation.

Generally, tartar presents an inorganic structure rich in Ca and P minerals, such as brushite, octacalcium phosphate, hydroxyapatite and whitlockite, and an organic matrix, mainly constituted by aerobic bacteria and yeast or just anaerobic bacteria.

Tonsilloliths occur most commonly on the crypts of the palatal tonsils and are composed of a mixture of organic matter, namely bacterial cells and epithelial debris, as well as inorganic material rich in Ca and P minerals such as hydroxyapatite. Volatile sulphur compounds produced by anaerobic bacteria are usually associated to these, in general, innocuous structures.

The current study involved the ultrastructure and chemical characterization of the calcified structures by scanning electron microscopy (SEM) combined with energy dispersive spectroscopy carried out with a JEOL JSM 7001F instrument with an INCA pentaFetx3 Oxford spectrometer operated at 15 kV. Higher resolution characterization has been performed by transmission electron microscopy (TEM) using a H8100 Hitachi instrument operated at 200 kV. SEM samples were prepared following metallographic procedures, whereas TEM samples were obtained following standard biological sample preparation procedures.

The results show that sialoliths present the most complex structure, with a central core surrounded by concentric layers, while tartar and tonsilloliths do not have a distinctive architecture (Figures 1 (a), 2 (a) and 3 (a). At higher magnifications, layered structures, as well as crystals could be found in sialoliths and tartar (Figures 1 (b) and 2 (b). Bacteria were common in all the calcified structures, although in tonsilloliths their abundance is higher (Figure 3 (b)). All calcifications have similar elemental constitution, with Ca and P, indicating the presence of calcium phosphates (Figures 1 (c), 2 (c) and 3 (c). Sulphur was also found associated with the organic matter in sialoliths and tonsilloliths, though the amounts found in the latter were much smaller than initially expected.

Based on the similarities found, new correlations between these calcification will be available. For instance, the mineralization process described in tartar can help understand the similar processes occurring in sialoliths and tonsilloliths, while the association between bacteria and sulphur in tonsilloliths can be a clue for their presence in sialoliths.

The work was carried out with financial support of the Portuguese Foundation for Science and Technology through PTDC/SAU-ENB/111941/2009 and PEst-OE/CTM-UI0084/2011 grants.

Nanoparticles in general (NPs) and/or nanomaterials offer remarkable opportunities in industrial production, daily consumables, medicine, biotechnology, electronics and numerous other important commercial and economical areas. Among all these areas, nanomedicine has opened novel treatments for problematic diseases such as viral, genetic, cancer, AIDS, etc. There is limited information available regarding translocation and distribution of NPs in the body and in the environment. Additionally, there is also need for more information on NPs toxicity. Recently has been demonstrated that physiological barriers such as pulmonary and gastro-intestinal tract are affected.

The main objective of this work is to use functionalized metal NPs, as emissive agent markers, assess their internalization in cells and evaluate toxicity to cells.

Using the emissive two probes synthesized in a one-pot reaction using fluoresceine as chromophore, several gold (Au), round shape, and silver (Ag) NPs (round and triangular shapes) were functionalized in organic media and water by Brust and Turkevish methodology, using tetraoctylammonium bromide (TOABr) as a common stabilizer and sodium borohydride as reducing agent. All has been characterized by UV-vis and emission spectroscopy, transmission electron microscopy (TEM) (Figure 1), and Light scattering. To study the route of internalization into the cell NP-complexes were injected intraperitoneally in fish (Carassius auratus). After 48 hour fish were sampled and sacrificed and liver and intestine processed for histology examination. Additional sub-samples were stored at – 80ºC for enzymatic analysis (glutathione-S-transferase and catalase). Blood was also collected from healthy non-injected fish, for leucocyte separation followed by incubation with the metal NPs and cell viability assays. The presence of emissive NPs in cells was examined by microscopy using a Leica microscope (ATC 2000) adapted for epifluorescence (EF).

The microscopy analysis showed that apparently both metal NPs were internalized by leucocytes and intestine cells (Figure 2a and 2b) but apparently not by hepatocytes. However, it is still to clarify if NPs internalization occurred in dead or dying cells only, with more permeable membranes, or also in living cells. Another possibility relates to the detection limits and resolution of the microscope used: the fraction of NPs entering is too low and not detectable with this type of equipment. No significant fluorescence was detected in controls. Viability assays showed higher mortality rates in leucocytes incubated with triangular Ag NPs suggesting that the type of metal and shape have influence in cell toxicity. In general, enzymatic assays indicate low oxidative stress for cells. However, GST results show significant (p > 0.05) differences in livers from fish injected with round Ag NPs. With respect to catalase, significant differences (p > 0.05) were detected in livers from fish injected with round Au NPs. Although the presented results are preliminary they suggest that functionalized NPs are able to penetrate cell membranes. On the other hand, the observed toxicity can be attributed to differences in shape and type of metal NPs.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/MAR/119068/2010 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.

Helicobacter pylori is a Gram-negative microorganism that grows on microaerophilic conditions and has only one known natural reservoir: the gastric mucosa. The infection by H. pylori is very common worldwide and this bacterium is associated with the development of gastritis, peptic ulcer gastric cancer or gastric Mucosa Associated Lymphoid Tissue (MALT) lymphoma. Although its natural habitat is the acidic gastric mucosa, H. pylori is considered to be a neutralophile. The bacterium survives brief exposure to pHs of <4, but growth occurs only at the relatively narrow pH range of 5.5 to 8.0, with optimal growth at neutral pH. Recently we have identified a prophage sequence (prophage phiHP33) in the strain B45, isolated from a patient diagnosed with gastric MALT lymphoma. This prophage revealed to be very difficult to induce. In fact, only few phage particles were observed on electron microscopy micrographs after exposure to UV radiation.

In the present work we have compared the exposure to UV and to acidic environment in the induction of the prophage into a lytic cycle. We have tested two strains, the strain B45 carrying the prophage phiHP33 and a clinical strain 1152, isolated from a patient with peptic ulcer, that was revealed to be negative for the presence of integrase gene (a prophage gene essential for genome integration of prophage) by PCR, as negative control. Since the H. pylori reservoir is the human stomach the exposition to acid is very common, and with this experiment we intended to test if acid can trigger a phage lytic cycle.

The induction using UV radiation has been previously described. For acid induction we have used a protocol adapted from Karita and Blaser. A 48 hours culture of H. pylori was grown in Brucella broth (Oxoid) supplemented with 10% of fetal bovine serum (Gibco) and 1% of Polivitex (BioMérieaux) in microaerophilic conditions at 37ºC. The liquid culture was centrifuged and the cell pellet ressuspended in citrate-phosphate buffer pH 6 and incubated 15 minutes, centrifuged again and ressuspended in citrate-phosphate buffer pH 3 and incubated for 30 minutes. After centrifugation the supernatant was recovered and incubated for 3 hours in phage precipitant (33% polyethylene glycol [PEG], 3M NaCl). After centrifugation at 10000 rpm for 10 minutes at 4ºC the pellet was ressuspended in phage buffer. These samples were analysed by transmission electron microscopy (TEM) after negative staining with 1% aqueous uranyl acetate, using a JEOL 100SX electron microscope.

For B45 strain the induction using UV radiation (previously reported in Lehours, 2011) and acid exposure produced similar results (Figure 1 and Figure 2) showing numerous phage-like particles of about 100 nm diameter, apparently lacking a tail, after UV or acid exposition, respectively. These particles were not observed in the control strain 1152. Currently we are analysing the samples using molecular biology techniques and fixation embedding followed by ultrathin sectioning for TEM analysis, to detect the presence of phages.

These preliminary results suggest that acid also appears to induce the H. pylori prophage phiHP33. However, since the number of phage particles observed is small, we can not rule out that the observed particles were released spontaneously. The exposition to the natural acidic environment of the human stomach may induce H. pylori prophage into a lytic cycle and to the propagation of phages among different H. pylori strains colonizing the same individual. Although highly speculative, transduction may be another form of horizontal gene transfer, which has not been described for this bacterium yet.

Financial support received from the Portuguese Science and Technology foundation under the contract PTDC/EBB-EBI/119860/2010.

Several theories have been put forward regarding the aetiology and pathogenesis of salivary calculi, although a comprehensive understanding of the nucleation and growth mechanisms involved in the formation of these structures is still lacking.

In general, sialoliths present one core partially or highly mineralized surrounded by concentric layers of organic and mineralized matter that alternate in succession following a chronologic sequence. The layers consist of fine mineralized strata intercalated with fine organic ones and threaded globular structures with variable degrees of mineralization.

The exact mechanism involved in the genesis of sialoliths remains largely unknown, theories defending an initial organic nidus or an initial precipitation of minerals, with subsequent deposition of organic and inorganic layers, can be found in the literature. Nevertheless, it remains object of discussion the etiologic factors responsible for the formation of the first nidus or the initial precipitation, since infection, inflammation of the gland, viscous nature of the mucous secretions or naturally existing sialomicroliths have all have been implicated.

Aiming at an exhaustive systematization of salivary calculi morphogenesis, their morphology has been studied by micro-computed tomography (bCT) and scanning electron microscopy (SEM). CCT studies were done on as-extracted dried samples using uCT SkyScan 1172 instrument with a 1.3 Megapixel camera, operated at the maximum available power of the source (10W). Radiographs acquisition was performed with a rotational step in the 0.70-1° range, until a maximum of 180º, with an exposure time in the 3.1-5 s range. Microscopy observations were carried out with backscattered electron (BSE) signals using a JEOL JSM 7001F operated at 15 kV, samples were previously prepared following metallographic procedures.

The submandibular and parotid calculi investigated presented similar growth patterns, which can follow either concentric (Figure 1) or perturbed-growth typologies (Figure 2), although in most situations a gradation between them has been found. Nevertheless, a single well-defined core constituted by material with low mineralization was frequently present, supporting the nucleation hypothesis of an initial organic nidus.

The combination of TCT with SEM enabled a comprehensive characterization of the sialoliths: (i) the former technique allowed for a precise localization of the core and other morphological features within the calculus volume, while (ii) investigation of details at higher resolution could be achieved with the latter method. However, due to the friable nature of the sialoliths, handling during sample preparation results often in material loss (compare (a) and (b) in both Figures).

The work was carried out with financial support of the Portuguese Foundation for Science and Technology through PTDC/SAU-ENB/111941/2009 and PEst-OE/CTM-UI0084/2011 grants.

Salicornia ramosissima J. Woods (Chenopodiaceae), included in the species aggregate S. europaea agg., is an annual succulent halophyte and one of the most salt tolerant plants, broadly distributed in the salt marshes and salt pans of Ria de Aveiro and in many others of the Iberian Peninsula. Salicornia L. has been used not only as a seasoned vegetable, salad and fermented food in coastal areas of Europe and Asia, but also as folk medicine for disorders such as constipation, obesity and diabetes. To corroborate this, the literature reports immunomodulatory, antioxidative, anti-inflammatory, anti-hyperlipidemic and antidiabetic effects. Moreover, some bioactive compounds from its aerial parts were recently isolated and identified, exhibiting also antioxidant and cytotoxic activities.

Factors such as the proper botanical identification, season and harvest site, extraction and purification methods, characterisation of the active constituents and the extract effects in a dose-and time-dependent manner are crucial to assess the therapeutic potential of herbal extracts. The aim of this study was to investigate the possible hepatic and renal effects of an ethanolic extract of S. ramosissima on mice.

Aerial portions of S. ramosissima were collected from a salt pan in Ria de Aveiro (Portugal). The ethanolic extract (50 mg/kg b.w.) was orally administered, during 3 weeks, to male ICR-CD1 mice, purchased from Harlan (Spain). Control group was also considered. Liver and kidney were collected and prepared for histology. Animal procedures were followed according to guidelines for ethics and animal care.

Central and portal vein congestion and severe hydropic changes of the hepatocytes were noted in the liver of exposed group, compared with controls (Figs. 1A, 1B). Renal profile of control group evidenced normal features (Figs.1C, 1E). However, significant histological alterations were found in the exposed group: cortical interstitial haemorrhages (Fig. 1D), inflammatory cell infiltration and tubular cell degeneration within medulla and cortico-medullary junction (Fig. 1F).

In conclusion, this pilot study has demonstrated considerable effects in the mouse metabolism of S. ramosissima ethanolic extract, with significant hepatic and renal lesions. Further studies are needed to correlate these data with isolated active constituents for a more reliable evaluation of the potential of this species.

Acknowledgements

Grants were provided by CICECO, QOPNA and CESAM.

The use of microreactors in (bio)chemical processes has been gaining relevance in the last decade. The low consumption of reagents, the possibility of continuous operation and the faster translation from lab- to production scale are some of the several advantages of these devices. The whole results in cost reductions in process development. Enzyme catalyzed reactions have proved to be an excellent alternative to the use of chemicals due to its ability to catalyze the most complex chemical processes under benign experimental and environmental conditions. In this way, enzymes may be crucial to the implementation of a much more sustainable chemical industry.

The present work is within such scope, using as model system the immobilization of invertase in glass (silica) microchannels, for the production of fructose syrups through sucrose hydrolysis. The immobilization of the enzyme was achieved through treatment of the substrate with a sequence of coatings (Figure 1). Activation of the inner surface of the microchannels with 3-aminopropyltriethoxysilane (APTES) was followed by the introduction of a spacer, glutaraldehyde. Lastly the enzyme solution was introduced in the presence of sodium cyanoborohydride, in order to enhance the stability of the support-enzyme binding.

The characterization of the coatings at each stage of the immobilization protocol was carried out to confirm the change of the microchannel surface. Such studies were performed using Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) analysis (Figure 1).

The results obtained, namely the shifts on the surface roughness corroborate that the several coatings were successfully applied and the enzyme immobilized. Moreover, the immobilization approach used proved to be highly effective, resulting on successful continuous use of the microreactor for a period of 30 days with roughly constant full conversion of a sucrose solution of 50g/l, at a flow rate of 7µl/minute (Figure 2).

Future work will involve a more extensive characterization of the several coatings by Quartz Crystal Microbalance which will be decisive to achieve a better comprehension of the coating phenomena and hence optimize the immobilization process.

The authors would like to thank Fundação para a Ciência e a Tecnologia, Portugal, for financial support through contracts under the program Ciência 2007 awarded to P. Fernandes, for the doctoral grant SFRH/BD/74818/2010 awarded to F. Carvalho and for the doctoral grant SFRH/BD/71990 /2010 awarded to Patrizia Paradiso.

A very rare subset of Epidermolysis bullosa simplex (EBS), termed “EBS superficialis” (EBSS), has been described in two families. EBS superficialis is a rare autosomal dominant form of suprabasal EBS in which blistering arises just below the stratum corneum.

The original report of EBSS was subsequently questioned by genetic analysis of one of the families that identified a mutation in a collagen gene responsible for dystrophic epidermolysis bullosa (DEB), thus implying that this entity is in fact a variant of DEB. Nonetheless, the existence of some form of EBSS can not be ruled out.

We performed a transmission electron microscopy study of a skin biopsy from a 36 year old woman suspect of having EBSS. A skin biopsy was sequentially fixed in 3% glutaraldehyde in sodium cacodylate buffer 0.1M pH 7.3, osmium tetroxide in the same buffer and uranyl acetate in acetate-acetic acid buffer 0.1M pH 5. Dehydration was performed in ethanol and embedding in an Epon-Araldite mixture. Thin sections contrasted with uranyl acetate and lead cytrate were observed and photographed in a JEOL 100SX electron microscope.

She had a few intact acral bullae in the neonatal period and superficial erosions and crusts in friction areas since then. Her one year-old boy, her older sister and the latter’s adolescent son had the same clinical picture. The patient does not display any symptoms expected for DEB.

We found profound alterations of the structure of the outer layers of the epidermis. Degenerative changes were seen in the cells of the upper layers of the stratum spinosum. Nuclei are lost and flattened cells made mostly of compressed tonofilaments lay below a cleft that develops between the compressed cells and the granular cell layer (Fig. 1). The granular cell layer is very reduced and represented by a few damaged cells that stay attached to the stratum corneum (Fig. 2).

The alterations do not seem to be related to collagen defects, and in the reported case the cleft develops in the altered upper region of the prickle-cell layer, just below the granular layer.

Hepatolithiasis (HL) or intrahepatic calculi is an uncommon condition in Western countries, with a prevalence under 1%. It is much more frequent in East Asia, reaching 20% in China and Taiwan, up to 50% of which show associated cholelithiasis. The authors report a European patient with primary HL of the left hepatic lobe, 12 years after cholecystectomy for acute calculous cholecystitis, now successfully treated after left lobe hepatectomy.

A 63-year-old caucasian man with a past medical history of hypertension, coronary artery disease, dyslipidaemia and urgent cholecystectomy 12 years ago (due to an acute calculous cholecystitis without ultrasonographic or perioperative evidence of bile duct dilation or choledocholithiasis, and with no cholangitis ever since) was now admitted to our ward following a 2-day course of fever (39ºC), epigastric pain, nausea, vomiting and tender right upper abdominal quadrant.

Bloodwork showed mild leukocytosis with neutrophilia and low platelets; elevated C-reactive protein, γGT, alkaline phosphatase and LDH; normal AST, ALT, bilirubin and amylase. Negative HBV, HCV and HIV serologies. CA19.9 was notably high (7500 U/mL), 200x above normal range (N≤37). A diagnosis of acute cholangitis was assumed on clinical and laboratory basis, despite the absence of jaundice. The patient began therapy with iv ceftriaxone and improved clearly over the next few days. Raised CA19.9 in the setting of acute cholangitis, however, forced us into further study directed at the possibility of underlying biliary or pancreatic malignancy.

Abdominal ultrasound disclosed multiple calcifications in the left lobe. CT displayed several left lobe intrahepatic bile duct dilations but no calcifications, thus suggesting intrahepatic cholesterol stones and cholangitis due to an obstacle before the hilum. MR-cholangiography was performed, showing marked dilation of the left intrahepatic bile duct and moderate dilation in the extrahepatic portion of the common bile duct (CBD). Neither exam showed any sign of cancer. Surgery was the therapeutic approach, and the patient underwent a left hepatectomy.

Intra-operative ultrasound study unveiled severe dilation of the left intrahepatic bile duct, which was filled with gallstones, but no choledocholithiasis and no tumour whatsoever. Both the surgical procedure and the post-operative period were uneventful.

Macroscopic liver section showed multiple dilated bile ductules containing fragile yellowish-green gallstones (Fig.1). Microscopy revealed cystic dilations of the left intrahepatic biliary tree packed with intraluminal gallstones (Fig.2), fibrosis and moderate chronic inflammatory infiltrate with lymphoid aggregates (Fig.3). Some areas of the epithelium displayed erosion and reactive changes. No biliary neoplasm was found.

Immunocytochemistry for angiogenesis or lymphocyte membrane markers are not available in our Hospital. Soon after surgery, CA19.9 values decreased abruptly and just 4 months after the left hepatectomy they were back within reference range.

Our working hypotheses were primary hepatolithiasis (HL), secondary HL and Caroli disease.

Secondary HL, due to stone migration from the CBD, was discarded because calculi in the CBD were absent in the cholecystectomy 12 years ago and in ultrasonographic studies both then and now. Caroli disease, a congenital condition characterised by intrahepatic bile duct dilation, was contradicted by the ultrasonography in 2000 showing normal bile duct dimension. Excluding these two entities made primary idiopathic HL our final diagnosis.

The patient is now asymptomatic, having returned to his normal life with no limitations.

This report showcases a rare entity known as primary hepatolithiasis, usually causing recurrent cholangitis in older patients of Asian descent but seldom seen in Europe. The steep increase in CA19.9 related to cholangitis, although previously reported, is also very uncommon. Another interesting aspect is the conspicuous inexistence, over 12 years, of recurrent episodes of acute cholangitis (a traditional finding in hepatolithiasis). These three features help to compose this most peculiar case.