Antti Vaheri

Clinical and Laboratory Manifestations of Sindbis Virus Infection: Prospective Study, Finland, 2002–2003

Sindbis virus (SINV) is widespread in Europe, Africa, Australia, and Asia, but clinical infection occurs as epidemics in a few geographically restricted areas. We recently proved, by virus isolation from patients, that SINV is the causative agent of Pogosta disease, a mosquito-borne rash-arthritis occurring as larger epidemics every seventh year in Finland. Altogether, 86 patients with serologically verified SINV infection were recruited to the present study during the 2002 outbreak. We now describe in detail the duration, incidence, and characteristics of different symptoms; hematological parameters; antibody kinetics; and presence of SINV in different tissue samples. SINV RNA detection or virus isolation from blood and/or skin lesions was successful in 8 patients. Immunoglobulin (Ig) M antibodies became detectable within the first 8 days of illness, and IgG antibodies became detectable within the first 11 days of illness. During the acute phase of Pogosta disease, the typical symptoms were arthritis, itching rash, fatigue, mild fever, headache, and muscle pain. The most notable finding was that, in 50% of the patients, joint symptoms lasted for >12 months.

Chlamydia pneumoniae Associated with Central Nervous System Infections

We identified 15 patients with serological evidence of current Chlamydia pneumoniae infection when we studied 263 patients with central nervous system infections among an adult population of 3 million in 1993. In 9 of the 15 patients no other associated or etiological agents were found. One patient died. Sequelae appeared in 7 patients. In neurological infections, C. pneumoniae may be more prevalent as an associated agent than appreciated, and adequate antibiotic therapy may be life-saving.

Characterization of the NF2 protein merlin and the ERM protein ezrin in human, rat, and mouse central nervous system.

The neurofibromatosis 2 (NF2) protein, merlin, is structurally related to the ERM (ezrin-radixin-moesin) protein family of membrane-cytoskeleton linkers and is mutated in nervous system tumors. Apart from tumor suppressor activity, merlins functions are poorly understood. We compared the localization and expression of merlin and ezrin in developing and adult brain and in brain-derived progenitor cells. Both proteins were widely but differentially expressed in human, rat, and mouse brain. In brain tissue and neuronal progenitor cell cultures merlin was predominantly found in neurons while ezrin was expressed in astrocytes. Merlin expression was seen from E11 in mouse embryos, whereas ezrin was present earlier. Both proteins were expressed in embryonic mouse neurospheres, where ezrin was specifically localized in filopodia of adherent neuronal progenitor cells. Subcellular analysis demonstrated ezrin in fine filopodial structures in astrocytes, while merlin was detected in neuronal synaptic junctions. The widespread expression of merlin in brain and its association with protein kinase A suggest a role for merlin in brain biology.

Arthritis and arthralgia three years after Sindbis virus infection: Clinical follow-up of a cohort of 49 patients

Sindbis virus (SINV) emerges as large human outbreaks in northern Europe every 7 years. Similar to many other alphaviruses, SINV is a mosquito-borne causative agent of a rash-arthritis. Previous reports suggest that in many alphavirus infections joint symptoms might persist for years. A prospective cohort of 49 patients was physically examined 3 y after verified acute SINV infection to reveal persistent joint symptoms. We carefully searched for a temporal association between the infection and current symptoms, and took into account other medical conditions. Sera were collected and analysed with enzyme immunoassays. Arthritis (swelling and tenderness on physical examination) was diagnosed in 4.1% (2/49) of the patients. Tenderness on palpation or in movement of a joint was found in 14.3% of the patients in the rheumatological examination, and an additional 10.2% complained of persisting arthralgia at the interview. Thus, 24.5% of the patients had joint manifestations attributable to the infection 3 y earlier. A positive IgM antibody response persisted in 3/49 of the patients; both patients with arthritis were in this group. As one-quarter of the patients were symptomatic 3 y after infection, it seems that persistent symptoms of SINV infection have considerable public health implications in areas with high seroprevalence.

Cell‐cell contact activation of fibroblasts increases the expression of matrix metalloproteinases

Background. We recently found that direct homotypic cell‐cell contacts between human dermal fibroblasts induce a novel form of cell activation leading to non‐apoptotic programmed cell death. As the major features of this process we identified massive induction of cyclo‐oxygenase‐2 and production of inflammatory prostaglandins. On the surface of the decomposing spheroids, activation of the major extracellular proteolytic cascade, plasminogen activation, associated with surface exposure of α‐enolase, took place. Aim. To further characterize pericellular proteolysis by cell‐cell contact‐activated fibroblasts we studied the role of the other major extracellular proteolytic system, matrix metalloproteinases (MMPs). Methods. MMP expression in fibroblast clusters and monolayers was compared using mRNA microarrays and immunoblot analyses. The activities of MMPs were confirmed using MMP inhibitors and caseinolysis. Results. In microarrays MMP‐1, ‐10, and ‐14 (MT1‐MMP) were induced 5.8‐, 106‐, and 5.6‐fold, respectively. These findings were confirmed by immunoblotting. Radial caseinolysis showed low level of proteolytic activity in spheroid‐conditioned media; ilomastat, a general inhibitor of MMPs, suppressed 50% of the proteolytic activity thus confirming it to be at least in part due to MMPs. A cocktail of tetracycline‐derived MMP inhibitors suppressed lactate dehydrogenase (LDH) release only 11%, and if combined with aprotinin 28%. Conclusions. Cell‐cell contact activation of fibroblasts induced MMP‐1, ‐10, and MT1‐MMP expression, suggesting similar signaling to that in inflammation and cancer.

Plasminogen activation in human leukemia and in normal hematopoietic cells.

The active process of pericellular proteolysis is central in tumor invasion, and in particular the essential role of the urokinase‐type plasminogen activator (uPA) is well established. uPA‐mediated plasminogen activation facilitates cell migration and invasion through extracellular matrices by dissolving connective tissue components. uPA, its receptor (uPAR) and plasminogen activator inhibitor‐1 (PAI‐1) are enriched in several types of tumors. The importance of proteolysis and especially plasminogen activation is less clear in hematopoietic malignancies than in solid tumors. However, patients with leukemia have an increased tendency to bleeding, not always attributable to thrombocytopenia, and tissue infiltration by leukemic cells, processes in which plasminogen activation may be involved. Several studies have indicated that plasminogen activators (PAs) are highly expressed by cultured leukemia cells. Furthermore, differing from adherent tumor cells, leukemic cells have an enhanced capacity to activate pro‐uPA and mainly the active form of uPA is released to culture medium. Ex vivo studies have shown that uPAR, uPA and its inhibitors can be found on the surface of normal blood cells and on the blast cell surfaces from patients with acute leukemia as well as from plasma samples. Elevated levels of PAs and their inhibitors have been detected in leukemic cell lysates. Few studies have tried to demonstrate a correlation between prognosis of leukemia and levels of plasminogen activators. More in vivo studies are needed to show, if any of the factors of the plasminogen activation process can be used as tools in subclassification or as markers for prognosis in leukemia. This review article will focus on the in vivo studies of plasminogen activation in leukemia and will present several in vitro findings on PAs in normal leukocytes and leukemic cell lines.

Procollagen-III in serum, plasminogen activation and fibronectin in bronchoalveolar lavage fluid during and following irradiation of human lung

In the search for predictors of late radiation-induced lung injury we studied procollagen type III peptide concentration (P-III-P) in serum as well as fibronectin and plasminogen activation in bronchoalveolar lavage (BAL) fluid during and following irradiation of human lung. The patients received either high-dose hemithorax irradiation for pleural mesothelioma (11 patients) or high-dose irradiation with individually shaped fields for non-small cell lung cancer (12 patients). The severity of radiation fibrosis was assessed clinically from CT scans 6 months and 12 months after treatment. Four scores were used: severe, moderate, mild, or normal. Radiological lung injury varied from severe (9 patients) to near absence of injury-normal (6 patients). Serum levels of P-III-P, when measured weekly during the 5-week period of radiotherapy or at several time-points after treatment, did not show consistent changes, nor did the levels correlate with the score for radiation fibrosis as assessed by CT scanning. Changes in fibronectin levels or in markers of plasminogen activation in BAL fluid did not correlate with the development of late lung injury. The levels of BAL fluid plasmin and plasminogen activator as assessed zymographically, but not the free net enzyme values, showed a tendency to be elevated in patients with severe radiation-induced lung injury, suggesting a possible role for inhibitors of the plasminogen activation cascade in the process of radiation-induced lung injury.

Molecular epidemiology of viral pathogens and tracing of transmission routes: hepatitis-, calici- and hantaviruses

BACKGROUNDnThe need to rapidly identify new therapeutic drugs and vaccines for clinically important viral infections has resulted in intensive study of the molecular properties of viruses. Modern molecular techniques have provided tools for tracing infections and studying the evolution of viruses. OBJECTIVE STUDY AND DESIGN: Two examples illustrating how modern molecular techniques can be used in clinical virology and molecular epidemiology (hepatitis and caliciviruses), and one example documenting their importance in basic research (hantaviruses) will be discussed.nnnRESULTS AND CONCLUSIONSnWater- and food-borne outbreaks caused by the faeco-orally spread hepatitis A virus (HAV) are common in areas lacking proper sanitation, but they are possible also in countries with low seroprevalence. In water epidemics, the sequence comparisons between the virus from patients and from water have been used successfully. Hepatitis B virus variants are clinically important and challenge the diagnostic tests and prophylactic measures. Some hepatitis C (HCV) genotypes appear to be associated with more severe pathology and others respond better to antiviral treatment. Nosocomial and occupational infections are not rare, and the source can be identified by phylogenetic analysis of nucleotide sequences obtained from the infected individuals. The overwhelming role of Norwalk-like caliciviruses (NLV) in adult diarrhoea and especially in food- and water-borne epidemics has become apparent during the last decade. Methods are under development for detecting these viruses, not only from patient samples and water, but also from other environmental samples (e.g. foodstuff and surface swabs). The analysis of the genetic variation and evolution of the Old World hantaviruses in their carrier rodents has shown that the extent of genetic diversity correlates with geographical distance. As a rule, phylogenetic relationships of hantaviruses resemble those of their rodent hosts, suggesting virus-host co-evolution. Exceptional host-switch events allow a study on still radiating hantavirus species. There is suggestive evidence that natural reassortant hantaviruses are involved in human infection.

Human herpesvirus-6 and -7 DNA in cerebrospinal fluid of facial palsy patients

Conclusions. Finding human herpesvirus (HHV)-7 and dual HHV-6A and -6B DNA in cerebrospinal fluid (CSF) of two facial palsy (FP) patients is intriguing but does not allow etiologic conclusions as such. HHV-6 or -7 DNA was revealed in 10% of the CSF samples tested from 70 immunocompetent adolescents and adults; a highly unusual result. How these findings are associated with the diseases they accompany remains to be defined. Objective. To determine whether herpes simplex virus (HSV)-1 and -2, varicella-zoster virus (VZV), HHV-6A, -6B, and -7, Epstein–Barr virus (EBV), and cytomegalovirus (CMV) DNA could be found in CSF of FP patients or controls. Subjects and methods. In all, 33 peripheral FP patients (26 idiopathic, 5 with herpesvirus infection, 1 puerperal, 1 Melkersson-Rosenthal syndrome) (34 CSF samples) and 36 controls (16 nonidiopathic FP, 7 hearing loss, 6 vertigo, 5 headache, 2 other) previously tested for HSV-1, VZV, and HHV-6 DNA by polymerase chain reaction (PCR) were tested with highly sensitive multiplex-PCR and an oligonucleotide microarray method. Results. One FP patient had HHV-7 DNA and another had HHV-6A and -6B DNA simultaneously. In the control group, one HHV-7, one HHV-6A, and three HHV-6B DNA-positive specimens were found.

Plasminogen activation in neurofibromatosis 2-associated and sporadic schwannomas

SummaryBackground. Schwannomas are usually benign tumours which occur sporadically or in association with neurofibromatosis 2 (NF2), an autosomal dominant disorder. Invasiveness and higher proliferative potential compared to sporadic tumours are features of NF2-associated schwannomas.n Method. We studied urokinase (uPA), tissue-type plasminogen activator (tPA), urokinase receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) expression by in situ hybridization and by immunohistochemisry in 14 NF2 and 15 sporadic patients with 34 schwannomas. uPAR and vitronectin immunohistochemistry were also studied. Three sural nerve specimens were included as Schwann cell controls.n Findings. Both schwannoma groups expressed prominent levels of uPA and tPA. Semiquantitative analysis of the in situ hybridization and immunoreactivity demonstrated that NF2 schwannomas expressed less PAI-1 at the mRNA level than sporadic schwannomas (score 1.63±0.41 vs. 2.05±0.75) and less total PAI-1 at the antigen level (score 1.55±0.66 vs. 2.07±0.56). PAI-1 was mostly in a free form in NF2 schwannomas compared to the sporadic counterparts (score 1.85±0.73 vs. 1.46±0.58), whereas there was less uPAR antigen in NF2 schwannomas than in the sporadic counterparts (score 1.18±0.49 vs. 1.68±0.56). Sural nerve Schwann cells did not express detectable level of PAI-1 and at the most a minor amount of tPA.n Conclusions. Schwann cells of tumour cell origin, both in sporadic and NF2 schwannomas, expressed elevated levels of plasminogen activators and PAI-1 compared to normal suralic nerve Schwann cells. Furthermore, there seemed to be an imbalance in the PA-PAI-1 system in NF2-associated schwannomas. Although our methods are more descriptive than quantitative, we suggest that the somewhat more aggressive behavior of NF2-associated schwannomas compared to sporadic schwannomas may be based on the local proteolytic activity.