The forward and reverse primers that we used to amplify the pro-IL-16 gene are 5′-CGG GAT CCA TGG ACT ATA GCT TTG-3′ and 5′-CGA CGT CGA CCT ATG AGT CTG CAG AA-3′, respectively. The forward and reverse primers

for amplifying the control GAPDH gene are 5′-CCG ATG CCC CCA TGT TTG TG-3′ and 5′-GGC CAT GCC AGT GAG CTT CC-3′, respectively. To measure the level of cell proliferation, 5 × 104 cells were suspended in growth medium together with a stimulator. After a 48-h incubation, MTS/PMS solution (Promega) was added, and the mixture was incubated for an additional 90 min at 37 °C. The absorbance was then measured at 490 nm using a SpectraCount™ ELISA reader (Packard Instrument Co., Downers Grove, IL, USA). Statistical analyses were performed using SigmaPlot™ (Systat Software, Chicago, IL, Y-27632 solubility dmso USA). Results are presented as means ± standard errors. An unpaired Student’s t-test was used to compare groups, and P values less than 0.05 were considered significant. We previously demonstrated that MHC class II molecules repress

resting B cell activation when they are cross-linked by an anti-MHC class II antibody. In this study, we used a functional learn more proteomics strategy to characterize the profiles of MHC class II-associated proteins dynamically involved in the regulation of resting B cell activation. Initially, MHC class II-associated proteins were enriched by immunoprecipitation, separated by 2-DE and identified through Q-TOF mass spectrometric analysis, as described in the materials and methods section. Our goal was to analyse proteins expressed

at high levels in a short period (15 min) after stimulation to focus on post-translational modifications of signalling molecules and to minimize potential fluctuations in levels of protein expression. We identified 10 known and unknown proteins that may have roles in cytoskeletal rearrangement, proliferation, intracellular signalling and metabolic regulation (data not shown). Among these proteins, pro-IL-16 drew our primary attention because it has been shown to act as a cell-cycle suppressor in T cells [18, 19]. Consequently, we investigated whether 4-Aminobutyrate aminotransferase pro-IL-16 is associated with MHC class II-associated resting B cell activation signalling. Densitometric analysis of the spots corresponding to pro-IL-16 in the gels showed that the level of pro-IL-16 was increased by LPS treatment of 38B9 resting B cells after 15 min and that the LPS-mediated increase was inhibited by co-treatment of cells with the corresponding anti-I-Ad MHC class II antibody (Fig. 1A, upper panel). When we checked the mRNA levels using RT-PCR with pro-IL-16-specific primers, we detected a similar pattern of pro-IL-16 transcript expression in cells treated with either LPS or LPS together with anti-MHC class II antibody (Fig. 1A, lower panel).

There were no flap losses, but four flaps (20%) developed congestion at the tip of the Selleckchem Lumacaftor flap that resolved without need for flap delay, leeching, or vasodilators. No patients developed complications with the donor site, and no patients underwent revisions. With a mean follow-up of 27.3 months (range: 19–38 months), all patients were pleased with their aesthetic outcomes and alive without recurrent disease. Conclusion:

The STAP flap is a pedicled perforator flap providing local “like” tissue that can be utilized for resurfacing of defects involving the anterior upper external ear with minimal donor site morbidity. © 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“Objectives/Hypothesis: The primary objective of the study was to determine the frequency of intraoperative vasopressor administration among patients undergoing free tissue transfer for head and neck reconstruction, and the secondary objective was to determine the impact of intraoperative vasopressor on free tissue transfer outcomes, including the impact of cumulative vasopressor dose and timing of intraoperative vasopressor administration. signaling pathway Study design/Methods: A retrospective review was performed of all patients undergoing free tissue transfer for head and neck reconstruction at the University Health Network between 2004 to 2008. Results:

From 2004 to 2008 inclusive, 485 patients underwent 496 free tissue transfers for head and neck reconstruction. The complete failure rate was 2.2% (11 of 485 patients). The partial failure

rate was 1.4%, and the operative take-back rate for venous congestion or arterial thrombosis was MAPK inhibitor 1.6%. This gave a total major flap complication rate of 5.2%, which was used as the primary free tissue transfer outcome measure. Of the 485 patients who underwent free tissue transfer, 320 (66.0%) received intraoperative vasopressor. Of these patients, the majority (97.5%) received phenylephrine and/or ephedrine. There was no significant relationship between receiving intraoperative vasopressor and major free flap complications, which were defined as complete failure, partial failure, or operative take-back for venous congestion or arterial thrombosis. Conclusion: Intraoperative vasopressors are used routinely in free tissue transfer for the reconstruction of head and neck defects. The use of intraoperative vasopressors does not appear to adversely affect free tissue transfer outcomes. © 2011 Wiley Periodicals, Inc. Microsurgery, 2012. “
“Biosynthetic guides can be an alternative to nerve grafts for reconstructing severely injured peripheral nerves. The aim of this study was to evaluate the regenerative capability of chitosan tubes to bridge critical nerve gaps (15 mm long) in the rat sciatic nerve compared with silicone (SIL) tubes and nerve autografts (AGs).

© 2010 Wiley-Liss, Inc. Microsurgery,

2011. “
“Ear amputation is a devastating injury characterized by a conspicuous deformity click here that is not easily concealed and can result in tremendous psychological trauma in addition to the physical insult. While numerous different approaches have been proposed, microvascular replantation is widely considered to deliver the best esthetic outcome. In this article, the authors report a case in which an unconventional perfusion pattern (i.e., arterialization of the venous system) was chosen, as intraoperative anatomic conditions precluded conventional vascular reconstruction. A 25-year-old male patient sustained a human bite resulting in subtotal amputation of his left ear. In the setting of an adequate arterial donor vessel, that is, branch of the posterior auricular artery, and a single suitable recipient vein (0.4 mm), the decision was made to perform an end-to-end arterio-venous anastomosis without the use of vein grafts. Medicinal leeches were applied postoperatively to provide for venous drainage. The ear survived and the patient was discharged after 14 days. To the best of our knowledge, this is first case of a subtotal ear amputation that was successfully

replanted by arterialization of the venous system without the use of vein grafts and with preservation of the superficial temporal vessels. © 2014 Wiley Periodicals, Inc. Microsurgery 34:657–661, 2014. “
“Background: The choice of recipient vessels is an important factor NVP-AUY922 research buy for successful head and neck reconstruction. Finding good recipient vessels for neck microsurgery can be difficult HA-1077 mouse after patients have undergone radiation therapy, previous neck dissection or developed neck infections due to pharyngocutaneous fistulae.

Thoracoacromial arteries and veins can be good alternatives to common recipient vessels in such patients. We reviewed the complications, advantages and disadvantages associated with using thoracoacromial arteries and veins as recipient vessels. Methods: We reviewed eight patients whose thoracoacromial arteries and veins served as recipient vessels for head and neck reconstruction between 2002 and 2009. Preoperative status, reconstruction method and operative outcomes with complications were evaluated. Results: Postoperative complications related to microsurgical anastomosis developed in two of the eight patients. One arterial and venous thrombosis developed in each patient. We considered that the arterial thrombosis was derived from a technical problem with the operation and the venous thrombosis was derived from postoperative external pressure. Conclusions: Thoracoacromial arteries and veins are good recipient vessels for patients who have undergone ablative or reconstructive surgery, radiation therapy, or have a neck infection due to complications.

Next, we found that removal of doxycycline from the drinking water after 4 weeks led to emigration of the CD45.1+CD19+GFP-high, hence miR-221-expressing cells from the BM within the next 4 weeks (Fig. 4A), CD19+sIgM+ B cells appeared in the spleen and, to a lesser extent, in the peritoneum (Supporting Information Opaganib Fig. 7). We conclude that miR-221-expression is responsible for residence and retention of the transplanted cells in BM. Upon termination of miR-221-expression, half of the transplanted mice did no longer retain

CD45.1+GFP+ cells in BM. Since we had found that CD19+sIgM+CD45.1+GFP+ mature B cells had developed in spleen and peritoneum in vivo in the presence of doxycycline it is likely that at least some of the CD19, sIgM pre-B cells had left the BM, had differentiated, and were now found as sIgM+GFP− B cells, no longer expressing miR-221 in spleen and peritoneum. In the other half of the

transplanted mice, CD45.1+ GFP-low-expressing cells could selleck screening library be detected in the BM even 4 weeks after the removal of doxycycline (Fig. 4A). This could be the result of an insertion of the miR-221 vector at a site in the genome that allowed continued low level-miR-221 expression, that is, GFP expression even in the absence of doxycycline, a condition that might allow pre-B cells to enter BM, but not to leave it again. Therefore, we subcloned the miR-221-transduced cell line in an attempt to separate the two types of miR-221-expressing GFP-expressing cells that were able to migrate to BM when miR-221 was expressed. Indeed,

a cell line could be derived which migrated to BM in all transplanted hosts in the 4-week-long presence of doxycycline induced miR-221-expression, and from where all CD19+CD45.1+sIgM−GFP+ pre-B cells disappeared when miR-221 expression was terminated by the subsequent 4-week-long removal of doxycycline (Fig. 4B). Other cell lines derived from these subcloning experiments either did not migrate to BM at all when miR-221 was expressed, or did not leave the BM, after expression of miR-221 was terminated. This suggests that induction of migration and termination of residence might depend on an optimal site of insertion of the miR-221 gene into the genome that allows optimal induction and Aldehyde dehydrogenase full termination of expression. We conclude that miR-221 expression controls the retention of pre-B cells in the BM. In order to test whether miR-221 overexpression is, indeed, responsible for the change in the migratory capacity of pre-B-I cells to the BM, we used a miR-221-complementary antagomir oligonucleotide to block the action of mature miR-221 in a sequence-specific fashion [24]. Doxycycline-induced, miR-221-expressing pre-B-I cells were loaded either with miR-221-specific antagomir or, as control, with unspecific, sequence-scrambled antagomir on the day of transplantation.

A moderate but statistically significant increase in CRP (P < 0.01)

and PCT (P = 0.01) was seen from the time of febrile neutropenia to 1–2 days later (Table 2). Moderate but statistically significant (P < 0.01) increases in the complement activation products C3bc and TCC were detected from the time of febrile neutropenia to 1–2 days later (Table 2), consistent with a moderate in vivo activation of complement during this period. Five patients were deficient for MBL (<60 μg/l), and five other patients had decreased values for MBL (219–326 μg/l), a prevalence of MBL variants that is the normal finding for a Caucasian population [8]. We found a modest but statistically significant (P < 0.05) change in 10 of the 17 cytokines measured (Table 2). Notably, three of them showed Kinase Inhibitor Library purchase a decline during the period, significant only for IL-5, though. The others showed very modest increases, indicating a lack Atezolizumab supplier of cytokine storm in these patients. IL-6, IL-8, IL-10, INFγ and TNFα correlated positively with each other both at the onset of febrile neutropenia, 1–2 days later and regarding the increases in the values of the cytokines. Unfortunately, there were too few patients with low MBL values in this population to make a statistical statement concerning a correlation with the cytokine pattern. The comparison of the patients who received tobramycin once daily

with those who received the antibiotic three times daily is presented in Table 3. We found a statistically significant higher increase

in the once-daily group compared with the three-times-daily group for PCT and for the following cytokines: IL-1β, IL-4, IL-6, IL-10, IL-12, GM-CSF, INFγ and TNFα (P < 0.05). The profiles of PCT, complement activation factors and cytokines suggested a mild inflammatory response in these lymphoma patients [16] undergoing high-dose chemotherapy with autologous stem cell support. The benign clinical course of the patients was in accordance with these findings. However, we were not able to make a conclusion as to our hypothesis. The results reflect only the situation in patients with a benign course of febrile neutropenia, and they say nothing about the inflammatory response in patients with a Gram-negative sepsis or a more 3-mercaptopyruvate sulfurtransferase severe course of febrile neutropenia. The CRP values showed a wide non-specific variation, reflecting neither the non-complicated clinical course nor the relatively low PCT and cytokine levels. Fifty of the 55 patients with paired blood samples had PCT values <0.5 μg/l, suggesting no bacterial infection [4]. As reference intervals have not been established for cytokines, the results in Tables 2 and 3 must stand on their own. Statistically significant median concentration increases were seen from the onset of febrile neutropenia to the drawing of the second sample for the cytokines, IL-1β, IL-4, IL-6, IL-7, IL-8, G-CSF, GM-CSF, INFγ and TNFα. There was on the other hand a statistically significant decrease in the IL-5 concentration.

1 M carbonate-bicarbonate HCS assay buffer, pH 9.6, coated onto a Nunc MaxiSorp® flat-bottom 96-well plate and incubated overnight at 4 °C. The plate

was washed with 0.05% PBS-Tween and blocked with 100 μL of 3% PBS-gelatin for 5 h at room temperature. Subsequently, 50 μL of twofold dilutions of the standard prepared in 1% PBS-gelatin, starting from a concentration of 32 ng mL−1, and 50 μL of the samples were added to the plate and incubated at 4 °C overnight. After washing, 50 μL of the secondary antibody diluted in 1% PBS-gelatin was added, and the plate was left at room temperature for 5 h, followed by the addition of 50 μL of 1:1000 streptavidin-peroxidase (KPL) prepared in 1% PBS-gelatin to each well and incubation at 37 °C for 30 min. The plates were developed with 100 μL well−1 of TMB (3,3′,5,5′-tetramethylbenzidine) substrate, the reaction was stopped by the addition of 100 μL well−1 of 0.2 M sulphuric acid, and A450 nm was measured. The ELISA for each cytokine was performed twice, and the samples and standards were tested in duplicates on each plate. Statistical analyses were performed using the graphpad Prism 4.0 software. The data generated from ELISAs were analysed by nonlinear regression, and interstrain comparison was performed by one-way anova. The role of surface-associated proteins and toxins of C. difficile in Angiogenesis inhibitor infection and serum antibodies to them in determining the outcome of infection has been

clearly demonstrated (Pantosti et al., 1989; Mulligan et al., 1993; Methisazone Péchiné et al., 2005a, b; Sánchez-Hurtado et al., 2008; Wright et al., 2008). Here, we demonstrate that toxins and surface-associated proteins from different C. difficile strains induce similar levels of production of pro-inflammatory cytokines by THP-1 macrophages. The SLPs, flagella and HSPs induced at 42 and 60 °C were extracted successfully from the five C. difficile strains, and the preparations were found to be free of endotoxin by the LAL assay. In the SLP extracts, two major bands were observed in preparations from all the five strains (Fig. 1a). As previously recorded, there was a wide variation in the molecular weights of the SLPs between

the different ribotypes (McCoubrey & Poxton, 2001; Spigaglia et al., 2011); strain 630, VPI 10463 and ribotypes 027, 001 and 106 were assigned S-layer types 5138, 5435, 5438, 5436 and 5037, respectively. In the flagella preparations, a prominent 39-kDa band (Delmée et al., 1990) was observed, which was the only band detected by Western blotting with rabbit antiserum prepared against whole UV-killed cells of C. difficile previously shown to react with flagella (McCoubrey & Poxton, 2001; Fig. 1b). A 58-kDa band was observed in HSP42 suggesting the presence of GroEL (Hennequin et al., 2001a; Fig. 1c), and three bands of approximately 66, 50 and 35 kDa were observed in HSP60 suggesting the presence of Cwp66 (Waligora et al., 2001; Fig. 1d).

This assay enables the potency of Treg cells from different HIV-1-infected groups to be compared by assessing their ability to suppress effector cells from healthy controls. Conversely, effector cells from different patient cohorts can be compared for their sensitivity to be suppressed by Treg cells isolated from controls. Using this assay, we provide unequivocal evidence that CD4+CD25+FoxP3+ Treg-cell potency in all chronic HIV+ subjects tested is comparable to controls tested in parallel, irrespective of their CD4+ T-cell count, virus load, disease stage or therapy status, using either a proliferation

assay or an IFN-γ intracellular staining (ICS) assay as a readout. The mechanism for the selective loss of effector cell proliferative capacity, but not Treg cell-suppressive potential, is presently unclear, especially as Treg cells Selumetinib in vivo appear to be

more readily infected than activated effector cells 15, 42, 43. The implication is that lower IL-2 expression, a hallmark of HIV infection 26, 27, accounts for loss in effector cell proliferation, without impacting the sensitivity of these cells to Treg-cell mediated suppression. This notion is supported by other data showing Treg suppression to be preserved in chronic HIV+ subjects and Simian Immunodeficiency Virus (SIV) models, selleck kinase inhibitor despite a fall in CD4+ T-cell count 4, 6, 8, 13, 14, 36. Furthermore, the preservation of Treg-cell potency in HIV infection is interesting, as Treg cells

are known to critically rely on IL-2 for expansion and function Cediranib (AZD2171) 44, 45 and may reflect threshold differences in IL-2 requirement for Treg and effector cell function. The second important aspect of this study is the observation that effector cell sensitivity to Treg-cell mediated suppression, using IFN-γ as a readout, is elevated only in chronic untreated HIV+ subjects but not progressor pre- and post-HAART. A previous report by Kinter et al. 13 also highlighted elevated suppression in lymph node Treg cells compared to peripheral blood, but did not establish if this is due to increased potency of patients Treg cells and/or an increased sensitivity of effector cells to Treg-cell suppression. A key question that arises from our data is whether increased effector cell sensitivity to Treg-cell suppression is linked to reduced IL-17 expression. Treg cell development is intimately linked to the counter-regulatory pro-inflammatory cytokine, IL-17, with Treg cells being negatively regulated by Th17 cells 31, 46. Evidence that this cannot be the sole explanation is provided. We demonstrate that effector cells from both chronic untreated and pre-HAART progressors are severely impaired in IL-17 expression. Indeed, progressors have significantly fewer IL-17+ cells than chronic untreated patients.

Typhi, can infect these mice and cause aspects of the pathology that is observed in human patients. However, with respect to the elicited human immune responses, more needs to be done to evaluate the immune competence of these models. While it has become clear thus far that isotype-switched humoral immune responses are difficult to achieve, cell-mediated T-cell immunity can be detected

in most of the investigated infections. In contrast to adaptive immune responses, 3-deazaneplanocin A innate immunity is still largely unexplored in most of these infectious settings and remains an interesting and promising topic for examination. Therefore, further studies are required to characterize in detail the immune competence of human reconstituted innate leukocyte populations. Moreover, apart from the evaluation of genetically modified pathogens, which the field is starting to explore, genetic modifications by viral check details transduction of transferred hematopoietic progenitor cells have to be established. In addition, more information on the donor variability of reconstitution in relation to genetic polymorphisms needs to be gathered. Furthermore, a set of antibodies that not only deplete reconstituted human leukocyte populations, but instead block distinct receptors, needs to be established. Finally, treatments that robustly induce secondary lymphoid tissues

in mice with reconstituted human immune system components would be of great value. While several additional ioxilan methodological developments are needed to improve the versatility of in vivo models of human immune responses, combining these efforts with recent and ongoing studies of infection and immunity in vivo promises to result in new preclinical models that are more predictive than current models for immune reactivity and therapy in patients. Work in our laboratory is supported by the National Cancer Institute (R01CA108609), Sassella Foundation (10/02, 11/02, and 12/02), Cancer Research Switzerland (KFS-02652–08–2010), Association for International Cancer Research (11–0516), KFSPMS and KFSPHLD of the University of Zurich, Vontobel

Foundation, Baugarten Foundation, EMDO Foundation, Sobek Foundation, Fondation Acteria, Novartis, and Swiss National Science Foundation (310030_143979 and CRSII3_136241). The authors declare no financial or commercial conflict of interest. “
“Macrophages and polymorphonuclear neutrophils are professional phagocytes essential in the initial host response against intracellular pathogens such as Mycobacterium tuberculosis. Phagocytosis is the first step in phagocyte-pathogen interaction, where the pathogen is engulfed into a membrane-enclosed compartment termed a phagosome. Subsequent effector functions of phagocytes result in killing and degradation of the pathogen by promoting phagosome maturation, and, terminally, phago-lysosome fusion.

[107] Immunohistochemistry localized p65 to CEC nuclei in Pkd1−/− kidney explants.[107] Similarly, Park et al. identified an unspecified phosphorylated NF-κB protein in CEC nuclei and in tubules surrounding the cysts of PKD2 mice and human ADPKD kidneys.[20]

Increased levels of phosphorylated and unphosphorylated NF-κB protein, and phosphorylated-IKKα/β were observed in PKD2 mice compared with wild-type mice, as well as increased levels of RAGE (receptor of advanced glycation end product, which is associated with renal inflammatory cell migration)[108] and s100a8 and s100a9 (inflammation-associated calcium binding proteins).[20, 109] In PKD2 mice, RAGE was located in CEC, and s100a8/a9 in CEC and interstitial areas proximate to inflammatory cells.[20] These data suggest that NF-κB activation is upregulated in human and animal models of PKD, and may be associated with increased inflammatory PARP inhibition mediators. Moreover, Qin et al. demonstrated that NF-κB inhibition modulated cystic disease, resulting in a three-fold decrease in histological cyst area.[107] NF-κB inhibition diminished the mRNA expression of three upregulated genes in PKD2 kidney explants: Wnt7a and Wnt7b, which are believed to be involved in polar cell polarity,[110] and Pax2, which is involved in embryonic nephron development.[107, 111, 112] NF-κB thus provides a promising

target for therapy, though further studies are required to characterize the effects, if any, of NF-κB on inflammation in PKD. Inflammation in PKD may be PS-341 clinical trial caused by abnormal regulation of the JAK-STAT pathway. Receptor binding of cytokines (e.g. IL-6 and interferon-γ), activates JAK proteins, which in turn activate STAT (signal transducer and activator of transcription) proteins, leading to gene transcription.[113] In vitro studies have shown that PC1 and PC2 are required for JAK1 and JAK2 activation,[114] and that Pkd1 regulates STAT3.[114] Therefore, Pkd1/2 mutations may promote inflammation by interrupting the control of JAK-STAT signalling. Furthermore, the JAK-STAT pathway is regulated by the suppressors of cytokine signalling (SOCS),[115] such as SOCS-1, which limits

the inflammatory activity of cytokines and macrophages.[116] Ribonucleotide reductase SOCS-1 knockout has led to the development of polycystic kidneys in mice,[117] but it is unknown whether this effect was mediated by inflammation or other facets of JAK-STAT signalling. Interstitial inflammation appears to correlate with disease progression in PKD. For example, heterozygous Han:SPRD rats display increased inflammatory cells at late stages of disease when there is severe interstitial fibrosis, proteinuria and extensive cystic expansion.[34] Given this, is it possible that inflammation induces cystogenesis? In some interventional studies, the amelioration of interstitial inflammation is accompanied by reduced cyst growth,[118, 119] though this does not prove causality.

, 1991; Roux et al., 1997). To amplify a 70-bp fragment targeting C. burnetii insertion element IS1111 (Denison et al., 2007), we applied a forward primer AAA ACG GAT AAA AAG AGT CTG TGG TT and a reverse Selleckchem Panobinostat primer CCA CAC AAG CGC GAT TCA T. The primers QHVE1 (TTC AGA TGA TGA TCC CAA) and QHVE3 (GAT

ATA TTC AGA CAT GTT), which amplified a fragment of variable size of the 16S–23S rRNA intergenic spacer (ITS) region, were used for confirmation of Bartonella (Roux & Raoult, 1995b). Borrelia was specified with 16S rRNA-encoding gene (Raoult et al., 1998). Primers Bf1 (GCT GGC AGT GCG TCT TAA GC) and Br1 (GCT TCG GGT ATC CTC AAC TC) were functional testing samples. The positivity of the amplification was confirmed by electrophoresis in a 1% agarose gel. The sizes of the PCR amplification products were determined by comparison with the molecular weight standard marker VI (Boehringer). If the amplification was positive, the PCR products were purified with Qiagen columns (QIAquick Spin PCR purification kit; Qiagen) and subsequently sequenced. Fifty serum samples were collected between days 1 and 45 after the onset of symptoms, selected from a prospective cohort study of severe affection after a tick or insect bite from 150 consecutive patients assigned with ‘unknown etiology’, obtained from various rural localities in the southeastern part of Slovakia (results

shown in Table 2, Fig. 3). After excluding viral infection (tick-borne ICG-001 concentration encephalitis, haemorrhagic fever), we tested them to examine the possibility of a bacterial origin of the disease. The selection for bacterial infections was done according to disease symptoms, epidemiological and clinical criteria, including myalgia and fever commencing no later than 10 days after a bite.

Twenty-seven (54%) female patients and 23 (46%) males of different age groups (from a 3-year-old child to an adult of 79 years) were included in the study. Forty-five patients were treated with antibiotics (tetracycline or doxycycline), one (no. 37) had a complicated course of illness (sarcoid myocarditis), and all of patients were hospitalized. All 50 serum samples were examined with the 22-antigen click here IFA (Tables 2 and 3). A multiple-antigen IFA was performed as previously reported (Fournier et al., 1998b), using three IgG and/or IgM titers of ≥ 1 : 25, ≥ 1: 50, ≥ 1 : 100 against any of the tested species. We detected 16 (32%) rickettsia-positive cases. IgG titers ≥ 1 : 100 in two cases were considered serological evidence of rickettsial infection, which was triggered by Rickettsia helvetica (no. 25, village Horča), and Rickettsia raoultii (no. 46, county of Lučenec). We identified sera from eight patients with a titer of ≥ 1 : 50 against R. helvetica [from the city of Levice (Nos 3, 5, 13), the villages of Kukučínov (no. 23) and Ondrejovce (no. 24) from the county of Levice, the villages of Mankovce (no.