Interaction of interstitial photodynamic therapy and interstitial hyperthermia in a rat rhabdomyosarcoma--a pilot study

Photodynamic therapy (PDT) involves the activation of photosensitizing drugs by light of appropriate wavelength. The photosensitive agent Hematoporphyrin Derivative (HPD) appears to be preferentially retained in malignant tumors; irradiation of HPD-containing tissue by light of appropriate wavelength (625 nm) and dose leads to (tumor) tissue destruction. The aim of this study is to achieve maximum tumor control probability with minimum normal tissue photosensitivity. In previous work from our laboratory it has been demonstrated that PDT has its fundamental effects on the tumor and normal tissue microcirculation. As it is well established that hyperthermia (HT) has its major effects in less well vascularized areas of the tumor, the combined modality of HT and PDT might prove to be advantageous. Moreover, suppression of sublethal damage repair by HT has been observed. To overcome the problem of poor light penetration into tissues and the high rate of recurrences following PDT with external irradiation, the combined effects of interstitial PDT with interstitial hyperthermia in a new line of animal experiments were studied in our laboratory. An experimental murine tumor (Rhabdomyosarcoma, type R-1) was transplanted in WAG/Rij rats and, after reaching an average diameter of 2 cm, the active component of HPD, that is Photofrin II, was injected intravenously in different dose schedules (5 mg/kg, 10 mg/kg). After 24 or 48 hrs the tumors were implanted with four flexible catheters, through which either light or heat could be applied. Light was obtained from an Argon-Dye laser system tuned to a wavelength of 625 nm at a dose rate of 75-100 mW per fiber to a dose level of 900 Joule from four linear light applicators. Heat (44 degrees C/30') was delivered by four 27 MHz radiofrequency antennas. Dose response relationships for PDT alone, HT alone and PDT combined with HT were established with cure as endpoint. This study showed that these two modalities, in the proper sequence and spacing, result in an augmented cytotoxicity on the tumor cells in vivo. With the combined modality treatment a cure rate of 41% (90 days) was obtained. As the implantation of flexible catheters is a well-known technique in radiation therapy practice, the potentiating effects of interstitial HT combined with interstitial PDT in solid tumors is very promising and clinical studies are warranted.     

Difficulties during removal of fluted femoral intramedullary rods

The difficulties that were encountered during removal of a fluted intramedullary femoral rod from six patients were reviewed. The mechanisms of failure included metal breakage at three different locations along the rod or the rod extractor. Three of the six rods were left in place due to difficulties in removal. The design of the fluted intramedullary rod appears to be the main cause of the problem.     

Urokinase in gastrointestinal tract bleeding

Selective urokinase infusion into the superior mesenteric artery allowed the accurate determination of the site of small bowel bleeding in a patient with recurrent lower gastrointestinal bleeding who bled despite resective surgery and who had negative findings on four angiograms. Fibrinolytic agents are useful in rare cases in which the need for successful and accurate diagnosis outweighs the risks of reactivating the bleeding.     

Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery

Urinary exosomes containing apical membrane and intracellular fluid are normally secreted into the urine from all nephron segments, and may carry protein markers of renal dysfunction and structural injury. We studied methods for collection, storage, and preservation of urinary exosomal proteins. We collected urine from healthy volunteers, added protease inhibitors, and stored urine samples at 4, -20, and -80 degrees C for 1 week or 7 months. Samples were thawed with and without extensive vortexing, and three fractions were isolated: urinary sediment, supernatant, and exosome fraction. Protein concentration, electrophoresis patterns, and abundance of seven exosome-associated proteins were measured. Exosome-associated proteins were not detected in sediment or supernatant fractions. Protease inhibitors prevented degradation of exosome-associated proteins. Freezing at -20 degrees C caused a major loss in exosomes compared to fresh urine. In contrast, recovery after freezing at -80 degrees C was almost complete. Extensive vortexing after thawing markedly increased exosome recovery in urine frozen at -20 or -80 degrees C, even if frozen for 7 months. The recovery from first and second morning urine was similar. The abundance of cytosolic exosome-associated proteins did not decrease during long-term storage. We concluded: (1) protease inhibitors are essential for preservation; (2) storage at -80 degrees C with extensive vortexing after thawing maximizes the recovery of urinary exosomes; (3) the difference between first and second morning urine exosome-associated protein was small, suggesting minimal protein degradation in the urinary tract/bladder; (4) urinary exosomes remain intact during long-term storage. These urine collection, storage, and processing conditions may be useful for future biomarker discovery efforts.     

Biomarker and drug-target discovery using proteomics in a new rat model of sepsis-induced acute renal failure

Sepsis is one of the common causes of acute renal failure (ARF). The objective of this study was to identify new biomarkers and therapeutic targets. We present a new rat model of sepsis-induced ARF based on cecal ligation and puncture (CLP). We used this model to find urinary proteins which may be potential biomarkers and/or drug targets. Aged rats were treated with fluids and antibiotics after CLP. Urinary proteins from septic rats without ARF and urinary proteins from septic rats with ARF were compared by difference in-gel electrophoresis (DIGE). CLP surgery elevated interleukin (IL)-6 and IL-10 serum cytokines and blood nitrite compared with sham-operated rats. However, there was a range of serum creatinine values at 24 h (0.4-2.3 mg/dl) and only 24% developed ARF. Histology confirmed renal injury in these rats. Forty-nine percent of rats did not develop ARF. Rats without ARF also had less liver injury. The mortality rate at 24 h was 27% but was increased by housing the post-surgery rats in metabolic cages. Creatinine clearance and urine output 2-8 h after CLP was significantly reduced in rats which died within 24 h. Using DIGE we identified changes in a number of urinary proteins including albumin, brush-border enzymes (e.g., meprin-1-alpha) and serine protease inhibitors. The meprin-1-alpha inhibitor actinonin prevented ARF in aged mice. In summary, we describe a new rat model of sepsis-induced ARF which has a heterogeneous response similar to humans. This model allowed us to use DIGE to find changes in urinary proteins and this approach identified a potential biomarker and drug target - meprin-1-alpha.     

Prevalence and Associations of tcpC,a Gene Encoding a Toll/Interleukin-1 Receptor Domain-Containing Protein,among Escherichia coli Urinary Tract Infection,Skin and Soft Tissue Infection,and Commensal Isolates

TcpC, a new Toll/interleukin-1 receptor domain-containing protein of uropathogenic Escherichia coli involved in the suppression of innate immunity, was found in 2008. The aim of the present study was to determine the prevalence of tcpC and its association with virulence factors and phylogenetic groups among strains from a collection of 212 E. coli isolates from urinary tract and skin and soft tissue infections and 90 commensal E. coli strains.Pathogenic microbes avoid host defenses using a wide array of virulence factors. Escherichia coli strains, even though they are common bacteria of the gut microbiota, can be important pathogens due to the possession of virulence factors (5). Recently, Cirl et al. (1) reported that they found TcpC, a new Toll/interleukin-1 receptor (TIR) domain-containing protein of uropathogenic E. coli that inhibits Toll-like receptor (TLR) and MyD88-specific signaling, thus impairing the innate immune response. They further reported that tcpC homologous sequences were present in about 40% of E. coli isolates from individuals with pyelonephritis, 21% of isolates from individuals with cystitis, 16% of isolates from individuals with asymptomatic bacteriuria, and only 8% of commensal isolates. Their results suggested that TcpC increases the severity of urinary tract infections (UTIs) in humans and provided the first unambiguous evidence that bacterial pathogens interfere with TLR signaling to survive and spread in the human host.The aim of our study was to determine the prevalence of tcpC among 212 extraintestinal E. coli isolates: 100 E. coli isolates from individuals with symptomatic UTIs, 10 E. coli isolates from individuals with asymptomatic UTIs, 102 E. coli isolates from isolates from individuals with skin and soft tissue infections (SSTIs), and 90 E. coli commensal isolates. In addition, we investigated the association of tcpC with the phylogenetic group (groups A, B1, B2, and D; E. coli strains causing extraintestinal infections are known to mainly belong to group B2 and, to a lesser extent, group D, while commensal E. coli strains belong to groups A and B1), as well as with other well-known virulence factors of extraintestinal pathogenic E. coli (ExPEC) strains (cytotoxic necrotizing factor 1 [cnf1], hemolysin [hlyA], P-fimbrial adhesins [papGIII and papGII], S fimbriae [sfaDE], Afa/Dr adhesins [afa/draBC], aerobactin [iucD], and uropathogenic strain-specific protein [usp]). To our knowledge, this is the first investigation of the prevalence of tcpC among E. coli strains causing SSTIs and of the association of tcpC with phylogenetic group as well as virulence factor genes among UTI, SSTI, and commensal E. coli isolates.The extraintestinal E. coli isolates examined in this study were from our previous studies of UTIs (10, 12-14) and SSTIs (9), while the 90 E. coli commensal isolates were isolated for the purposes of this study. The commensal E. coli isolates were isolated as lactose-positive colonies on MacConkey agar plates from the feces of healthy individuals. Indole, methyl red, Voges-Proskauer, and citrate tests were performed to ascertain that the species detected were E. coli. The strains investigated were cultivated in Luria-Bertani medium or agar. Cell lysates of all 302 E. coli isolates were prepared (7) and used in the PCRs. Amplifications were performed in an automated thermal cycler (UNOII; Biometra, Göttingen, Germany) in a 25-μl reaction mixture containing template DNA (5 μl of boiled lysate), 10 pmol of forward and reverse primers (Table ​(Table1),1), 0.2 mM deoxynucleoside triphosphate mixture, 0.625 U Taq DNA polymerase, and 2.5 mM MgCl₂ in 1× PCR buffer (Fermentas, Vilnius, Lithuania). The amplification schemes were based on previous amplification protocols (Table ​(Table1).1). For amplification of the tcpC sequence, the following amplification scheme was employed: 1 cycle of denaturation at 94°C for 4.5 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 1 min. The amplification was concluded with an extension program of one cycle at 72°C for 5 min. Fisher''s exact test (two-tailed; http://www.langsrud.com/fisher.htm) and the Bonferroni correction were used to analyze the data. The threshold for statistical significance after the Bonferroni correction was set at a P value of <0.05. The PCR revealed that 49 (23%) of the pathogenic strains studied harbored the tcpC sequence: 23 (21%) of our UTI E. coli isolates (21 isolates [21%] from individuals with symptomatic UTIs and 2 isolates [20%] from individuals with asymptomatic UTIs) and 26 (25%) of our SSTI E. coli isolates. The prevalence of tcpC was much lower among commensal E. coli isolates, only 7 (8%), as was found in a recent study by Cirl et al. (1). Comparison of the prevalence of tcpC among the UTI isolates of the two studies was not possible, as we could not obtain data on the type of symptomatic UTI (cystitis, pyelonephritis), and furthermore, the number of asymptomatic UTI isolates was too small (n = 10) to be statistically relevant. As seen from Table ​Table2,2, strong statistical correlations were found between the presence of tcpC and the B2 phylogenetic group, as well as between the presence of tcpC and the presence of cnf1, hlyA, papGIII, sfaDE, and usp among UTI isolates, as well as commensal strains. Among the SSTI isolates, statistically significant associations were found only between the presence of tcpC and the presence of cnf1, hlyA, and usp. As ExPEC strains mainly belong to the B2 phylogenetic group, these correlations and the higher virulence scores of the tcpC-encoding strains are not surprising. Interestingly, when the UTI and SSTI isolates were compared, major differences were observed. While the prevalence rates of tcpC sequences were similar in both groups, 21% among UTI isolates and 25% among SSTI isolates, suggesting an important role of TcpC in UTIs as well as in SSTIs, P values establishing significant correlations were higher among UTI isolates than among SSTI isolates. The differences between the UTI and SSTI E. coli strains observed are most likely due to differences in pathogenic mechanisms; nevertheless, the possession of TcpC seems to be an important factor in establishing UTIs and SSTIs. As the bowel flora is a reservoir of ExPEC, it is not surprising that tcpC was also found to be significantly associated with the B2 phylogenetic group among commensal strains. Our results suggest that even though E. coli strains able to induce disease outside the gastrointestinal tract are collectively designated ExPEC (11), it could be worthwhile to consider strains from different sites or syndrome-specific pathotypes separately.

TABLE 1.

Sequences of primers used in this study

Imaging acute renal failure with polyamine dendrimer-based MRI contrast agents

Acute renal failure (ARF) induced by sepsis has a high mortality but lacks effective treatments. To develop novel therapies we must diagnose renal injury early and accurately in septic patients and identify any additional insults such as nephrotoxic drugs and ischemia. In this short review we describe our experience using MRI with dendrimer-based contrast agents in mouse models of ARF. This technique can diagnose early renal injury before serum creatinine is elevated, distinguish different ARF etiologies, track drug therapy and predict outcome. As an ARF biomarker, MRI with dendrimer-based contrast is a promising technique deserving further development.     

Signature profiles of CMV-specific T-cells in patients with CMV reactivation after hematopoietic SCT

Depletion of cellular immunity as a consequence of conditioning before allogeneic hematopoietic SCT (HSCT) frequently results in CMV reactivation, which may in turn lead to life-threatening infections and require timely antiviral treatment. We have investigated the functional signatures of CMV-specific CD4+ and CD8+ T-cells in 191 samples from 118 individuals. We compared healthy donors with both patients with high and undetectable viral loads, and those who controlled and did not control their CMV reactivations. Polychromatic flow-cytometric measurements of CD154 (CD40L), intracellular cytokines (IFNγ, IL2) and a degranulation marker (CD107a) allowed us to assess the functional status of various T-cells simultaneously. We found that dual IFNγ/IL2-producing CD8+ T-cells were present in patients controlling their CMV reactivations but absent from non-controllers. CD8+ T-cells that produce only IFNγ were the most abundant subtype, but they most likely represent non-protective memory cells. Distinct functional signatures were examined by hierarchical clustering, and this revealed that, unlike polyfunctional CD8+ T-cells, CD8+ T-cells that produce IFNγ alone were not functioning in concert with other subsets. In conclusion, our study revealed functional signatures that may be useful for immune monitoring, and it could change the interpretation of previous studies that assessed only IFNγ.