An outbreak of hepatitis A in Roma populations living in three prefectures in Greece.

An outbreak of hepatitis A virus (HAV) infection affected Roma populations living in three prefectures of northeastern Greece. Between July and November 2007, 124 cases were reported. We carried out investigations to characterize the pathogen, to identify the source of infection and the route of transmission. Using the RT-PCR technique, HAV strains of the same genotype were detected in all sera from a subset of patients with acute disease. These showed more than 99.8% identity, suggesting a common source. A questionnaire was also completed to collect clinical and epidemiological information. The outbreak affected mainly Roma children aged <10 years. An inspection of Roma settlements showed that poor sanitary conditions were associated with the HAV outbreak.

Bacteremia caused by Pantoea agglomerans and Enterococcus faecalis in a patient with colon cancer.

A case of bacteremia caused by Pantoea agglomerans (P. agglomerans) and Enterococcus faecalis in a male patient with colon cancer is reported. He was successfully treated with vancomycin and meropenem. The clinical isolate was identified as P. agglomerans by 16S rRNA gene sequencing.

Primary bacteraemia caused by Rhizobium radiobacter in a patient with solid tumours.

A case of Rhizobium radiobacter primary bacteraemia in a patient with solid tumours is reported. Corticosteroid therapy and diabetes mellitus were the predisposing factors. The patient was treated successfully with amikacin and piperacillin/tazobactam. The clinical isolate was identified as R. radiobacter by 16S rRNA gene sequencing. Phytopathogenicity tests and a PCR assay demonstrated that the organism was not a plant pathogen.

Central line-related bacteraemia due to Roseomonas mucosa in a neutropenic patient with acute myeloid leukaemia in Piraeus, Greece.

A case of central venous catheter-related bacteraemia due to Roseomonas mucosa in a neutropenic patient with acute myelogenous leukaemia is reported. The patient was successfully treated with amikacin and piperacillin-tazobactam. The clinical isolate was identified as R. mucosa by 16S rRNA gene sequencing.

Phylogenetic aspects of the complement system.

During evolution two general systems of immunity have emerged: innate or, natural immunity and adaptive (acquired), or specific immunity. The innate system is phylogenetically older and is found in some form in all multicellular organisms, whereas the adaptive system appeared about 450 million years ago and is found in all vertebrates except jawless fish. The complement system in higher vertebrates plays an important role as an effector of both the innate and the acquired immune response, and also participates in various immunoregulatory processes. In lower vertebrates complement is activated by the alternative and lectin pathways and is primarily involved in the opsonization of foreign material. The Agnatha (the most primitive vertebrate species) possess the alternative and lectin pathways while cartilaginous fish are the first species in which the classical pathway appears following the emergence of immunoglobulins. The rest of the poikilothermic species, ranging from teleosts to reptilians, appear to contain a well-developed complement system resembling that of the homeothermic vertebrates. It seems that most of the complement components have appeared after the duplication of primordial genes encoding C3/C4/C5, fB/C2, C1s/C1r/MASP-1/MASP-2, and C6/C7/C8/C9 molecules, in a process that led to the formation of distinct activation pathways. However, unlike homeotherms, several species of poikilotherms (e.g. trout) have recently been shown to possess multiple forms of complement components (C3, factor B) that are structurally and functionally more diverse than those of higher vertebrates. We hypothesize that this remarkable diversity has allowed these animals to expand their innate capacity for immune recognition and response. Recent studies have also indicated the possible presence of complement receptors in protochordates and lower vertebrates. In conclusion, there is considerable evidence suggesting that the complement system is present in the entire lineage of deuterostomes, and regulatory complement components have been identified in all species beyond the protochordates, indicating that the mechanisms of complement activation and regulation have developed in parallel.

Cloning and purification of the rainbow trout fifth component of complement (C5).

To gain further insight into the evolutionary history of the complement proteins C3, C4, and C5 we have now cloned the fifth component of complement from a rainbow trout (Oncorhynchus mykiss) liver cDNA library; this is the first report of C5 cloning in a species other than human and mouse. The deduced amino acid sequence of a partial cDNA clone (2.25kb), representing approximately 44% of the coding sequence, showed 60 and 58% similarity to human and mouse C5, respectively. To validate the molecular information derived from the cloning we developed an improved purification protocol. Mass spectrometric analysis of C5 tryptic digests yielded peptide signals that matched theoretical protein sequence derived from the partial cDNA. Northern blot analysis of RNA from various tissues showed the presence of a single mRNA transcript in trout liver and Southern blot analysis indicated that the gene coding for C5 is present as a single copy in the trout genome. The presence of C5 in trout suggests that C3, C4, and C5 must have diverged before the appearance of teleost fish.

Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity.

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5'-hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.

Cloning and structure of three rainbow trout C3 molecules: a plausible explanation for their functional diversity.

We have previously identified and characterized three distinct trout C3 proteins (C3-1, C3-3 and C3-4) that differ in their electrophoretic mobility, glycosylation patterns, reactivity with monospecific C3 antibodies, partial amino acid sequence and binding to various complement activators. To study the structural elements that determine the observed functional differences, we have cloned and sequenced the three C3 isoforms. Comparison of the deduced amino acid sequences showed that the sequence identity/similarity of C3-3 to C3-4 is 76/81%, whereas those of C3-3 and C3-4 to C3-1 are 55/67% and 54/67%, respectively. It is interesting that the beta-chain of C3-4 contains two insertions of 65 (residues 504-569) and 23 amino acids (residues 123-146), while the beta-chain of C3-1 contains a 14-amino acid insertion (residues 143-157). The C3 convertase cleavage site (Arg-Ser) is conserved in the three trout isoforms; however, the factor I cleavage sites are Arg-Ala (for C3-1 and C3-4) and Arg-Thr (C3-3) instead of Arg-Ser at position 1281 of human C3, and Arg-Thr (C3-1, C3-3) instead of Arg-Ser for C3-4 at position 1298 of human C3. Of special interest is the absence of the His(1126) and Glu(1128) (human C3 numbering) from C3-4 and of Glu(1128) from C3-3. These residues are thought to play an important role in determining the binding specificity of the thioester-containing proteins. Accordingly, we postulate that the distinct binding reactions of the trout C3 isoforms with various complement activators could be due at least in part to the observed changes in the His and Glu residues.

Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles.

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.

Complement diversity: a mechanism for generating immune diversity?

Unlike mammalian species, several cold-blooded species have been shown to possess multiple forms of complement components. The multiple forms of C3 characterized in several fish species can bind with different specificities to various complement-activating surfaces. Here, Oriol Sunyer, Ioannis Zarkadis and John Lambris explore the possible advantages conferred by having multiple forms of individual complement proteins in a single organism.

Cloning, structure, and function of two rainbow trout Bf molecules.

The factor B (Bf) and C2 complement genes are closely linked within the MHC class III region and are thought to have arisen by gene duplication from a single gene encoding an ancestral molecule; the animal phyla in which this duplication event took place is unknown. Two teleost fish, (zebrafish and medaka fish) have each been shown to possess only a single molecule that shows an equivalent degree of similarity to mammalian Bf and C2. In contrast, here we present the characterization of two factor B molecules (Bf-1 and Bf-2) in another teleost fish (the rainbow trout) that are about 9% more similar to mammalian factor B than C2, yet play a role in both alternative and classical pathways of complement activation. The full lengths of Bf-1 and Bf-2 cDNAs are 2509 and 2560 bp, respectively, and their deduced amino acid sequences are 75% identical. Both trout Bf genes are mainly expressed in liver and appear to be single-copy genes. The isolated Bf-1 and Bf-2 proteins are able to form the alternative pathway C3 convertase and are cleaved (in the presence of purified trout C3, trout factor D, and Mg2+ EGTA) into Ba- and Bb-like fragments in a manner similar to that seen for mammalian factor B. The most remarkable feature of trout Bf-2 is its ability to restore the hemolytic activity of trout Bf-depleted serum through both the alternative and classical pathways; whether Bf-1 possess similar activity is unclear at present.

cDNA cloning of the translocon associated protein beta-subunit in the chick cerebellum.

The 'translocon associated protein' is a tetrameric complex residing in the translocation sites, in which nascent polypeptides pass through the endoplasmic reticulum membrane. The beta subunit of this complex is a single spanning membrane protein, as deduced from cDNAs deriving from canine or human epithelial tissues. We have isolated and analysed a cDNA clone of the beta subunit from chick nervous tissue, namely cerebellum. Its deduced protein sequence is 91% homologous to both the canine and the human protein sequences, showing that the molecule is highly conserved.

Granulocyte-macrophage colony-stimulating factor improves immunological parameters in patients with refractory solid tumours receiving second-line chemotherapy: correlation with clinical responses.

In this report, we studied the immunorestorative properties of subcutaneously administered granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with refractory solid tumours receiving second-line chemotherapy. Such patients exhibit abnormal immune responses in vivo and in vitro and, therefore, it was of interest to examine the effect of GM-CSF-induced immunomodulation on clinical response. We examined patients with primary malignant carcinomas (head and neck, n = 10; urogenital tract, n = 17; penis n = 6; colorectal, n = 8) who were treated with carboplatin (JM8), 300 ng/m2 on days 1 and 22, leucovorin (LV), 200 mg/m2 plus 5-fluoracil (5-FU), 500 mg/m2 on days 8, 15 and 29 and four cycles of daily injections with placebo or GM-CSF, 300 micrograms/day on days 3-6, 10-13, 17-20 and 24-27. Peripheral blood was collected from the patients one day after the end of each of the four-cycle injections with placebo or GM-CSF, namely on days 7, 14, 21 and 28. Peripheral blood mononuclear cells (PBMC) were tested in the autologous mixed lymphocyte reaction (AMLR) and for natural killer (NK) or lymphokine-activated killer (LAK) cell activity. Cytokine levels in serum were measured by immunoenzymatic (ELISA) assay. A total of 21 patients received a four-cycle regimen with GM-CSF (Group 1) and 20 were similarly treated with placebo (Group 2). All received standard chemotherapy as outlined above. Before GM-CSF treatment, all patients exhibited increased serum levels of interleukin-1 (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha), IL-6 and prostaglandin E2 (PGE2) and decreased serum levels of IL-2. Cellular immune responses (AMLR, NK- and LAK-cytotoxicity) were also low in all patients. Five patients from Group 1 had a PR (partial response), 2 patients had CR (complete response), and 14 patients had stable disease. Seven patients from Group 2 showed progressive disease, 3 had a PR and 10 had stable disease. All immune parameters were significantly improved during treatment in Group 1 but remained unchanged or even deteriorated in Group 2. Administration of GM-CSF during treatment of cancer patients with conventional chemotherapeutic drugs results in a marked potentiation of deficient cellular immune responses in vitro and a change towards normalisation of cytokine serum levels. The results reported herein support the use of GM-CSF as immunopotentiator during chemotherapy, but more patients must be studied before definite conclusions can be drawn.

Differentiation-dependent changes in human trophoblast expression of decay-accelerating factor are modulated by 3',5' cyclic adenosine monophosphate.

OBJECTIVE: Decay-accelerating factor (DAF) is a complement regulatory protein that prevents complement-mediated cell lysis. Its expression in human trophoblasts depends on their anatomic location and gestational age. The mechanisms that govern the regulation of its expression in trophoblasts are not understood. The objective of the present study was to investigate the effects of trophoblast differentiation and cyclic adenosine monophosphate (cAMP) on DAF expression. METHODS: Human trophoblasts were isolated from chorionic villi, cultured, and harvested at timed intervals for total RNA extraction and Northern analysis. Expression of DAF was also assessed by immunocytochemistry. In some cultures, the trophoblasts were exposed to the cAMP agonists 8-bromo-cAMP and Sp-cAMPs or the antagonist Rp-cAMPs. RESULTS: Expression of DAF mRNA increased as the cells differentiated in culture, with the 2.2-kb transcript of membrane-bound DAF appearing first. As differentiation proceeded, the 1.5-kb transcript became the predominant mRNA form. Exposure of the cells to Rp-cAMPs delayed this process; 8-bromo-cAMP accelerated it. Sp-cAMPs selectively up-regulated the 2.2-kb mRNA transcript. Immunocytochemistry confirmed the effects of differentiation on DAF protein expression. CONCLUSION: Expression of DAF in human trophoblasts is dependent on the state of cell differentiation, and cAMP is an intracellular modulator of this process. This effect may be mediated through alternative processing of DAF mRNA in its 3'UT region, which in turn affects mRNA stability.

Local immunotherapy with interferon-alpha in metastatic pleural and peritoneal effusions: correlation with immunologic parameters.

Interferon-alpha (IFN-alpha) has been found to exert multiple enhancing effects in the immune response in vitro, IFN-alpha has been also used in clinical trials with variable response rates. The aim of the present study was to assess the effectiveness of IFN-alpha in the treatment of 25 patients with malignant pleural or peritoneal effusions caused by lung, and metastatic breast and ovarian cancer. Clinical responses were correlated with a) the ratio of malignant effusion (ME)-associated tumor cells to ME-associated mononuclear cells (MEMNC), b) MEMNC-derived cytotoxic responses against autologous or allogeneic tumor targets, and c) major histocompatibility complex (MHC) antigen expression on tumor cells. After partial drainage of pleural or peritoneal fluid, the patients were allocated to receive 10 million units of IFN-alpha by intrapleural or intraperitoneal injection at weekly intervals. The treatment was terminated if the malignant effusion disappeared or the patients had received four to six consecutive procedures. None of the patients received concomitant systemic chemotherapy or radiation therapy. MEMNC and tumor cells were isolated by centrifugation on discontinous percoll density gradients. Cytotoxic and phenotypic profiles of MEMNG were analyzed before and after treatment with IFN-alpha. An improvement was observed in patients with increased ratios of tumor cells to malignant effusion-associated mononuclear cells (MEMNC) in the effusions. In the same patients MEMNC were overpopulated by CD8+ T lymphocytes. In this group of patients the administration of IFN-alpha was associated with 25% complete response and 75% partial response rates. In contrast only 17% partial responses were achieved in patients whose effusions had decreased tumor cell to MEMNC ratios. The immunomodulation induced by IFN-alpha in vivo was also tested. Thus in a group of 6 patients, treatment with IFN-alpha resulted in the induction of CD8+ cell-mediated lysis against autologous tumor cells which was associated with PR (two patients). Natural killer (NK)-cell activity, and MHC class I antigen expression on effusion-associated tumor cells were also enhanced during treatment, but were not correlated with the outcome of the therapy since similar findings were also observed in the 4 non-responders. Local infusions of IFN-alpha provide an effective alternative treatment for malignant effusion in patients with lung, breast, and ovarian cancer. Increased ratios of tumor cells to MEMNC and the presence of CD8+ T lymphocytes within the malignant effusions may play an important role in the outcome of such a treatment with IFN-alpha but more patients need to be studied for definite conclusions.

Multiple forms of complement C3 in trout that differ in binding to complement activators.

In all other species analyzed to date, the functionally active form of complement component C3 exists as the product of a single gene. We have now identified and characterized three functional C3 proteins (C3-1, C3-3, and C3-4) in trout that are the products of at least two distinct C3 genes. All three proteins are composed of an alpha-and a beta-chain and contain a thioester bond in the alpha-chain. However, they differ in their electrophoretic mobility, glycosylation, reactivity with monospecific C3 antibodies, and relative ability to bind to various surfaces (zymosan, Escherichia coli, erythrocytes). A comparison of the partial amino acid sequences of the three proteins showed that the amino acid sequence identity/similarity of C3-3 to C3-4 is 87/91%, while that of C3-3 and C3-4 to C3-1 is 51.5/65.5% and 60/73% respectively. Thus, trout possess multiple forms of functional C3 that represent the products of several distinct genes and differ in their ability to bind covalently to various complement activators.

DNA haplotype heterogeneity of beta-thalassaemia in Greece: feasibility of prenatal diagnosis.

We have carried out DNA haplotype analysis of 69 beta-thalassaemia patients in Greece and 42 of the parents using seven standard polymorphic sites. Our data show a high degree of heterogeneity of the chromosomal background in which beta-thalassaemia occurs in Greece, suggesting a high degree of heterogeneity in the beta-thalassaemia mutations involved. Haplotype I is found here to represent 45% of total beta-thalassaemia mutations detected, a proportion well below the 67% reported in earlier studies with Greek-American patients. Nine different haplotypes are detected and the ones carrying beta(+) mutations are the majority, including those which are linked to beta(+) mutations associated with a thalassaemia intermedia phenotype, and which constitute 11% of all haplotypes. One of these haplotypes (---- ) has never before been reported to occur in non-Africans, whether in beta thal or beta A chromosomes, and it is found here to be of African origin rather than the product of recombination. In 21 families haplotype analysis showed that prenatal diagnosis for a second child was feasible in 81% of the cases. Use of the AvaII-psi beta polymorphic site as well as the seven standard ones brought this proportion up to 90%.