First report of Dientamoeba fragilis infection explaining acute non-specific abdominal pain.

Dientamoeba fragilis is now considered a potentially emerging gastrointestinal pathogen in both developing and developed countries. We first report an autochthonous case of D. fragilis infection in Greece. A 49-year-old female with acute non-specific abdominal pain required emergency surgical admission for active observation and repeated assessment. To the best of our knowledge, this is the first reported case of acute unexplained abdominal pain finally attributed to D. fragilis infection using microscopic and molecular methods.

Treatment of canine leishmaniosis with aminosidine at an optimized dosage regimen: a pilot open clinical trial.

Leishmaniosis due to Leishmania infantum (Syn: L. chagasi) is one of the most common diseases of dogs in Mediterranean countries and also has zoonotic potential. The aim of this study was to evaluate the efficacy of an optimized dosage regimen of aminosidine for the treatment of canine leishmaniosis (CanL) in terms of clinical remission, restoration of clinicopathological abnormalities, evolution of antibody titer, lymph node and bone marrow parasitic density and of PCR-based parasitological cure. Twelve non-uremic dogs without proteinuria, presenting clinical signs of CanL were included in the study. The diagnosis was confirmed by serology, microscopy and PCR of lymph node and bone marrow samples. Aminosidine was administered subcutaneously at the dose of 15 mg/kg body weight, once daily, for 21 consecutive days. A partial remission of the clinical signs, amelioration of clinicopathological abnormalities such as anemia, lymphopenia, hyperproteinemia, hyperglobulinemia, and reduced albumin/globulin ratio and reduced lymph node and bone marrow parasitic density were witnessed, although parasitological cure was not achieved. Since data are not supportive enough for the use of aminosidine as an alternative treatment, a large-scale controlled clinical trial using this optimized dosage regimen of aminosidine is warranted to compare efficacy against currently used drugs.

Malaria in Greece, 1975 to 2010.

Malaria, which was endemic in Greece in the past, was officially eliminated in 1974. Since that time and up to 2010, a number of imported cases (ranging from 19 to 76) have been annually reported. The total number of reported laboratory-confirmed cases between 1975 and 2010 was 1,419. Plasmodium falciparum was identified in 628 (44%) of these cases, while P. vivax was found in 524 (37%). Of the total cases, 1,123 (79%) were male (ratio males vs. females: 3.78). Age was only available for 490 cases, of which 352 (72%) belonged to the 18-40 year-age group. Of the 382 malaria cases reported from 1999 to 2010 for which the region/country of acquisition was known, 210 (55%) were from Africa and 142 (37%) from Asia. The massive introduction of economic migrants, in the period from 1990 to 1991 and from 2006 onwards, mainly from countries where malaria is endemic, resulted in the appearance of introduced sporadic cases. In Peloponnese, Central and East Macedonia, Thrace and East Attica, mosquitoes of the genus Anopheles (e.g. Anopheles sacharovi, A. superpictus and A. maculipenis) that can act as plasmodia vectors are abundant and during the summer of 2011, 27 P. vivax cases were reported in Greek citizens residing in the agricultural area of Evrotas in Lakonia and without travel history. As further P. vivax malaria cases occurred in the Lakonia and East Attica areas in 2012, it is becoming urgent to strengthen surveillance and perform integrated mosquito control that will help eliminate the potential risk of malaria reintroduction and reestablishment.

Determination of CD4+ and CD8+ T cells in the peripheral blood of dogs with leishmaniosis before and after prolonged allopurinol monotherapy.

Canine leishmaniosis (CL) is a common systemic parasitic disease that is endemic in many Mediterranean countries including Greece. The immune reaction to the parasite is critical to the outcome of the infection and the response to treatment. Some studies have shown a reduction of circulating CD4+ T cells and of the CD4+/CD8+ ratio in dogs with CL and these changes normalised following treatment with meglumine antimoniate or amphotericin B. Allopurinol is used as a monotherapy for the chronic treatment of CL. The aim of the present study was to determine the circulating CD4+ and CD8+ T lymphocyte numbers and the CD4+/CD8+ ratio in 19 dogs diagnosed with CL before and after prolonged allopurinol monotherapy (18 months). A significant decrease in circulating CD4+ T cells was observed in dogs with CL before treatment. Prolonged allopurinol monotherapy improved the number of circulating CD4+ T cells, but did not restore their number to within the normal range.

Molecular detection of Leishmania infantum in wild rodents (Rattus norvegicus) in Greece.

Leishmania infantum and Leishmania tropica are the species responsible for visceral leishmaniasis and cutaneous leishmaniasis respectively. In Greece, both diseases are endemic. The dog is considered the main reservoir of L. infantum, whereas the role of other animals for both L. infantun and L. tropica is unknown. Spleens from wild Rattus norvegicus, live trapped in Greece, were examined for the presence of Leishmania parasites by PCR. Out of 16 samples examined, only one was found positive for L. infantum with scant amount of parasitic DNA present. This is the first documented case of detection of L. infantum in R. norvegicus in Greece. The results of this preliminary study indicate that R. norvegicus is unlikely to be a reservoir for Leishmania parasites in Greece.

Molecular characterization of the Anopheles maculipennis complex during surveillance for the 2004 Olympic Games in Athens.

Specimens belonging to the Anopheles maculipennis complex were collected as larvae or resting adults from May 2003 to November 2004 in the area of the Athens 2004 Olympic Rowing Centre in Schinias, Attiki, Greece, and identified by morphological and molecular analyses. Of the 201 specimens collected, 199 were found to be Anopheles sacharovi Favre and two were An. maculipennis Meigen s.s. on the basis of similarity to published sequence data for the rDNA internal transcribed spacer (ITS2) region and the mitochondrial cytochrome c oxidase I gene (COI). Sequence data from a number of specimens were obtained for both genes and compared with corresponding GenBank data derived from diverse geographical areas. A high degree of homology in ITS2 sequences was found in both species, ranging from 99.5% to 100% in An. sacharovi and 99.4% to 100% in An. Maculipennis, with no intraspecific variation in either of the two species in our study. The degree of homology in the COI sequences was 94.8-99.8% in An. sacharovi and 95.0-99.8% in An. maculipennis. The 522-bp fragment produced a rather high degree of intrapopulation polymorphism for An. sacharovi, generating nine different haplotypes, five of which were singletons. Intraspecific variation for these sequences ranged from 0.2% to 1.4%, but was much lower (0.77%) for the two An. maculipennis sequences. These findings represent the first characterization of the An. maculipennis complex in the area of Schinias.

Genotyping of pathogenic Acanthamoebae isolated from clinical samples in Greece--report of a clinical isolate presenting T5 genotype.

Amoebae belonging to the genus Acanthamoeba are potentially pathogenic to humans, causing mainly amoebic keratitis. Pathogenic ability of the 15 known Acanthamoeba genotypes is under investigation. We report that four out of five cases of amoebic keratitis studied in Greece, present T4 sequence type, while the remaining one presents T5 sequence type (Acanthamoeba lenticulata), which is the second most frequent genotype found among environmental samples. Thus, it is confirmed, for the first time to our knowledge, that A. lenticulata can cause keratitis. However the reason that it is under represented in clinical samples compared to environmental ones is unknown.

A single-step, PCR-based method for the detection and differentiation of Plasmodium vivax and P. falciparum.

The ability to detect and differentiate between Plasmodium falciparum and P. vivax is of great importance for the routine laboratory diagnosis of malaria, donor-blood screening and epidemiological studies. Most PCR-based methods for the discrimination of these two species require nested protocols or an additional hybridization reaction, leading to high labour costs and long turn-around times. A simple, time-effective and yet sensitive and specific technique, based on a multiplex PCR, has now been developed for the simultaneous detection and differentiation of P. falciparum and P. vivax in blood samples. Compared with the 'gold standard' of microscopy, this method had a sensitivity and specificity of 100%, with a detection limit of just one P. falciparum or three P. vivax parasites/microl blood.

Cytokine serum levels, autologous mixed lymphocyte reaction and surface marker analysis in never medicated and chronically medicated schizophrenic patients.

A number of immunological parameters were studied in 82 DSM-III-R diagnosed schizophrenic patients (53 first drug-naive and 29 medicated chronic patients) as well as 62 healthy blood donors. The serum levels of interleukin-2 (IL-2), interleukin-1 beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha) were measured and correlated with cellular immunity, as assessed by the autologous mixed lymphocyte reaction (AMLR). T lymphocyte subsets were also examined. The above immune parameters were reassessed in a subgroup of 11 first-episode, drug-naive patients 1month after neuroleptic medication. IL-2 serum levels were significantly lower, and IL-1beta and TNF-alpha were significantly higher in schizophrenic patients compared with healthy donors (P<0.001); no significant difference was observed between the two patient groups (medicated and not medicated). Abnormal cytokine serum levels were associated with decreased AMLR responses in vitro. Increased percentages of activated CD4+ and CD16+ natural killer cells, as well as cells expressing ICAM-1 adhesion molecules and IL-2 specific receptors, were detected in the patients. Immunophenotype studies revealed a higher percentage of cells expressing IL-2 receptors in medicated chronic schizophrenic patients compared with drug-naive patients. The abnormal cytokine production in vivo, along with the low AMLR responses in vitro, and the high percentage of activated CD4+ lymphocytes presented in this study suggest alterations in the immune system of schizophrenic patients (medicated or not medicated) consistent with immune activation.

A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces.

The detection of and differentiation between Entamoeba histolytica and Entamoeba dispar are of great importance, both for diagnosis and for epidemiological studies. Most PCR-based methods for the discrimination of these two species employ complex procedures for DNA extraction and require different protocols for E. histolytica and E. dispar, leading to relatively high expenditure, labour costs and turnaround times. A simple, rapid, cost-effective and yet sensitive and specific multiplex PCR technique has now been developed for the simultaneous detection and differentiation of E. histolytica and E. dispar in faecal samples. The detection limit is 200 trophozoites of E. dispar or 1000 trophozoites of E. histolytica/g stool sample. The sensitivity of the assay remains practically unchanged, even in the presence of 20,000 trophozoites of the other species/g stool sample. Thus, this technique may also easily reveal mixed infections, without the danger of misdiagnosis caused by one strain displacing the other in culture.

Increased generation of autologous tumor-reactive lymphocytes by anti-CD3 monoclonal antibody and prothymosin alpha.

Anti-CD3 monoclonal antibody (mAb) activates in vitro peripheral blood mononuclear cells (PBMC) to lyse a variety of tumor cell lines in a non-major histocompatibility-complex(MHC)-restricted manner [subsequently referred to as anti-CD3-activated killer (AAK) cytotoxicity]. Prothymosin alpha (ProTalpha) is a biological response modifier that exerts its effects primarily on mononuclear cells, especially when these cells' effector functions are impaired. In this study, we report that ProTalpha enhances the AAK cytotoxicity in PBMC from healthy donors. This effect was more profound with cancer patients' PBMC, which were deficient in their ability to respond with enhanced AAK cytotoxicity upon in vitro stimulation with anti-CD3. Thus, cancer patients' PBMC, activated with a combination of anti-CD3 and ProTalpha, exhibited increased AAK activity and efficiently lysed both autologous tumor and Daudi targets. The ProTalpha effect on PBMC was demonstrated to involve stimulation of adhesion molecules (CD2, CD18, CD54, CD49f) and CD25 expression, up-regulation of perforin mRNA transcription, increased numbers of perforin-positive (+) cells and elevated production of interleukin-2 (IL-2), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). Moreover, effector CD8+ and CD56+ cells pretreated with anti-CD3 and ProTalpha contained high cytoplasmic perforin levels and increased expression of IL-1beta- and TNFalpha-specific receptors. The induction of autologous-tumor-reactive CD8+ and CD56+ lymphocytes in anti-CD3-activated PBMC by ProTalpha provides an alternative protocol aimed at the improvement of clinical results in cellular adoptive immunotherapy of cancer.

Improved detection of Dirofilaria repens DNA by direct polymerase chain reaction.

Diagnosis of human infection by Dirofilaria repens, depends mainly on microscopic evaluation of tissue cross-sections and the macroscopic characteristics of the worm. Tissue degeneration and/or poor specimen preparation practices however, often render many cases of subcutaneous dirofilariasis elusive to such morphological diagnostic approaches. The early PCR protocols, developed to satisfy these complex diagnostic needs, failed to amplify dirofilariae DNA from formalin preserved material. To overcome these difficulties, we developed an improved PCR protocol using a set of primers designed to amplify a rather stable, highly repetitive D. repens-specific genomic DNA target. We report the performance of this protocol with a large variety of dirofilariae infected DNA specimens, including those extracted from formalin preserved biological material for up to 20 days. Our findings support its potential application to routine clinical diagnosis.

Induction of anti-tumour lymphocytes in cancer patients after brief exposure to supernatants from cultures of anti-CD3-stimulated allogeneic lymphocytes.

The present study investigated the ability of supernatants collected from cultures of healthy donor-derived peripheral blood mononuclear cells (HD-PBMCs) stimulated with anti-CD3 monoclonal antibody (MAb) (allogeneic CD3 supernatants; ACD3S) to induce, upon brief exposure, tumour-reactive cytotoxic lymphocytes in cancer patients' PBMCs. ACD3S enhanced natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity. ACD3S contained increased levels of interleukins (IL) 1, 2, 6, 7 and 12, as well as of granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma-interferon (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). MAbs against these cytokines significantly reduced the ACD3S-induced cytotoxicity. ACD3S-induced cytotoxicity was not inhibited by anti-CD4, CD8 and MHC class I MAbs, but was markedly reduced in the presence of MAb against CD18. In contrast to HD-PBMC, ACD3S derived from cancer patients' lymphocytes exhibited lower levels of the above-mentioned cytokines and exerted reduced biological activity. In conclusion, ACD3S are able to activate, upon short-term incubation, tumour-reactive lymphocytes from cancer patients' PBMCs that lyse a variety of tumour targets, including autologous tumours. ACD3S contain high levels of certain cytokines that positively influence the induction of autologous tumour-reactive lymphocytes. Such supernatants can be collected easily from healthy donors and stored until use in clinical trials for adoptive cellular therapy of cancer. They may also be indicated in the construction of cytokine cocktails that have the ability to induce anti-tumour cytotoxicity.

Production and characterization of a monoclonal antibody against epirubicin.

Epirubicin is an anthracyclinic antibiotic that has been increasingly used in the treatment of a variety of malignancies. A hybridoma producing monoclonal antibody (MAb) against the drug was obtained by cell fusion. The MAb is of the IgM isotype and has an affinity constant of 1.4 x 10(-7) M. Inhibition analysis showed that the antibody recognizes an epitope related to the C 4'-hydroxyl group in the amino sugar moiety, distinguishing epirubicin from the closely related doxorubicin. Since the precise mechanism of anthracycline action as well as its immunomodulating effects are still under scrutiny, powerful tools for their study are clearly needed. Moreover, this MAb can be useful in monitoring the levels of epirubicin in treated patients, as well as for the construction of bispecific antibodies in tumor-targeting immunotherapy.

Induction of tumor-specific T lymphocyte responses in vivo by prothymosin alpha.

We have recently reported that administration of ProT alpha to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor alpha [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunothera 38:281]. In this report we demonstrated that ProT alpha, when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumor-specific CD8+ cytotoxic T lymphocytes (CTL). The ProT alpha-induced CD8+ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2Kd allelic product could inhibit the cytotoxic response. Mice receiving only ProT alpha developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving ProT alpha and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The ProT alpha-induced CD8+ CTL activity was regulated by ProT alpha-induced L1210-specific syngeneic CD4+ cells. This was shown in two different ways: first, CD8(+)-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4+ cells and, second, CD4+ cells from mice that had received both ProT alpha and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8+ cells from mice that had received only L1210 cells. Our data suggest that ProT alpha is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since ProT alpha may prove to be useful in clinical protocols aimed at cancer immunotherapy.

An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry.

The use of the chromium-release assay to determine cytotoxicity of effector against target cells has various limitations mostly due to the inherent properties of the radioactive substance. We have developed an improved flow cytometric method that is able to measure cytotoxicity, based on two fluorescent dyes. Calcein-AM, a non-fluorescent substance which is intracellularly converted to the green fluorescent calcein by esterase activity in viable cells, is initially used to stain target cells. After incubating targets with effectors for 2 h, ethidium homodimer-1, a red DNA stain non-permeable to viable cells, is added. Dead target cells are distinguished by their double (green-red) staining. Data analysis is performed by gating the regions of living target, dead target and living effector cells, based on appropriate controls. Non-specific events are subtracted from the dead target region and the ratio of specific dead target events to total target events is expressed as percent cytotoxicity. The method is used to quantify natural killer (NK) and lymphokine-activated killer (LAK) activities against the human K562 and Daudi cell lines and the murine YAC-1 and L1210 cell lines respectively, as well as cell-mediated lympholysis (CML) exerted by tumor-infiltrating lymphocytes (TIL) against autologous and allogeneic human breast cancer tumor cells. The method is fast, reliable and correlates well with the standard 51Cr-release assay.

Human chromosome 19 confers the CD2/E rosette receptor phenotype in interspecific cell hybrids.

Interspecific cell hybrids between Chinese Hamster Ovary (CHO) and phytohaemagglutinin (PHA) stimulated human T lymphocytes were purified by preparative rosetting with sheep red blood cells (SRBC). The hybrid cell clone used in the present study consisted of cells containing a complete set of the 20 CHO chromosomes and one extra human chromosome, No 19. Hybrid cells constitutively expressed high levels of human CD2 surface receptor and formed multilayer rosettes with SRBC and human erythrocytes. In addition to CD2 they produced low levels of a small number of human extracellular proteins. These findings suggest that the factor(s) responsible for CD2 expression are produced by the hybrid and that genes responsible for CD2 expression are located on chromosome 19. However, the present work cannot exclude that material of chromosome 1, where the CD2 gene has been assigned previously, is integrated somewhere in the hybrid karyotype. Further work is needed to clarify this point.