AC133+ umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis

Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133+ cells (2-4 x 10(4)/ml), mononuclear cells (MNC, 1-2 x 10(6)/ml) and AC133negative cells (AC133(neg), 2-4 x 10(4)/ml) in stroma-free 8 d liquid culture (fetal bovine serum-supplemented Iscove's-modified Dulbecco's medium (IMDM) with either 'K36EG'[c-Kit ligand, interleukin 3 (IL-3), IL-6, erythropoietin, granulocyte colony-stimulating factor] or 'TPOFL' (thrombopoietin, Flt-3 ligand). Cell enumeration, apoptosis assay and AC133/CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133+ cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133(neg) populations (K36EG FI: 15.04 +/- 5.46; TPOFL FI: 8.59 +/- 2.92, P < 0.05). After 8 d, populations lacking AC133+ cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0.01). Also, when compared with K36EG, 8 d TPOFL-expanded AC133+ cells encompassed a significantly higher percentage of AC133+ and CD34+ early HSPC (K36EG: 20.50 +/- 2.36; TPOFL: 47.00 +/- 7.69; P < 0.05). In conclusion, TPOFL synergism demonstrated the potential for AC133+ HSPC ex vivo expansion inducing self-renewal, early HSPC maintenance and promoting cell survival status.     

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (K_m, 13.6 μM) and pregnenolone (K_m, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-³H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-³H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.     

Bacterial DNA in synovial fluid cells of patients with juvenile onset spondyloarthropathies

OBJECTIVE: To identify bacterial DNA in synovial fluid cells of patients with active juvenile onset spondyloarthropathy (SpA). METHODS: The main group of study constituted 22 patients with juvenile onset SpA. In addition, five patients with adult onset SpA and nine with rheumatoid arthritis (RA) were studied. Polymerase chain reaction (PCR) with either genus- or species-specific primers was performed on synovial fluid cells to detect DNA sequences of Chlamydia trachomatis, Yersinia enterocolitica, Salmonella sp., Shigella sp., Campylobacter sp. and Mycobacterium tuberculosis. The presence of antibacterial antibodies in sera and synovial fluid was also determined by enzyme-linked immunoassay. RESULTS: The synovial fluid of nine patients with juvenile onset SpA, three with adult onset SpA and one with RA contained bacterial DNA. Five juvenile onset SpA samples had DNA of one single bacterium; two juvenile onset SpA and three adult onset SpA had DNA of two bacteria and two juvenile onset SpA had DNA of three bacteria. Overall, Salmonella sp. DNA was detected in seven synovial fluid samples, Shigella sp., Campylobacter sp. and M. tuberculosis were found in four samples each, and C. trachomatis was found in two. The bacterial DNA findings correlated with neither diagnosis nor disease duration. One RA synovial fluid had DNA of Campylobacter sp. Neither serum nor synovial fluid antibacterial antibodies correlated with DNA findings or clinical diagnosis. CONCLUSION: In this study, single and several combinations of bacterial DNA were identified in the synovial fluid of patients with long-term undifferentiated and definite juvenile onset SpA and adult onset SpA. Of relevance is that bacterial DNA corresponds to bacteria producing endemic disease in our population.     

Hepatic and splanchnic perfusion and oxygenation after transjugular intrahepatic portosystemic shunt.

OBJECTIVE: in patients with cirrhosis, transjugular intrahepatic portosystemic shunt (TIPS) decreases the pressure in the portal vein by rerouting nearly all the portal blood flow to the systemic circulation. This may lead to hypoperfusion of the liver and worsening function. Our aim was to investigate whether TIPS actually reduced hepatic and splanchnic perfusion. METHODS: we studied 25 patients who required placement of a TIPS (20 for variceal bleeding and 5 for refractory ascites). We evaluated the clinical condition, laboratory results, blood velocity in the portal vein and hepatic artery by echo-Doppler ultrasonography, systemic hemodynamic-oxygenation status and hemodynamic-oxygenation status in the portal and suprahepatic veins before TIPS, 15 min after the procedure, and 30 days later. Hepatic and splanchnic perfusion were evaluated as the arteriovenous difference in O2 content and as the O2 extraction rates in the hepatic and splanchnic territories. RESULTS: TIPS induced an immediate decrease in portal pressure, a significant increase in systemic hyperdynamic state, and an increase in blood flow velocity in the portal vein and hepatic artery. Thirty days after the procedure these changes persisted, although they were somewhat attenuated. Although splanchnic and liver perfusion were not changed 15 min or 30 days after TIPS, there was a slight tendency toward a decrease in liver perfusion during follow-up. CONCLUSIONS: TIPS increased the hyperdynamic state in the systemic side. However, portal blood shunting did not change liver or splanchnic perfusion.     

Primary actinomycosis of the abdominal wall. Description of 2 cases and review of the literature

We report two cases of isolated abdominal wall actinomycosis and review 18 previously reported cases to further characterize the clinical findings and the therapeutic management of this syndrome. This diagnosis would be advocated in patients with a palpable abdominal mass of subacute appearance with a previous history of digestive medical illness, diabetes, abdominal surgery, or prolonged IUD use. In contrast with other actinomycosis locations, remarkable data were a more elevated mean age of patients; a female predominance; a prevalent location of mass in abdominal lower left quadrant; and a shorter duration of symptomatology before to diagnosis. The CT is the first choice for imaging study and percutaneous needle aspiration would be recommended for definite diagnosis. The long-term antibiotic therapy, with or without percutaneous drainage, is the first treatment choice because is very effective and made unnecessary a more invasive surgical management. The prognosis is excellent with adequated treatment.     

Suppression of experimental autoimmune encephalomyelitis using estrogen receptor-selective ligands

Estrogens have been shown to modulate disease activity in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis. Consistent with these findings, the severity of disease is reduced in pregnant women with multiple sclerosis when levels of estrogens are high. Estrogens bind to two known estrogen receptors (ER), ERalpha and ERbeta. The relative contribution of these receptors to estrogen-mediated suppression of EAE was explored using ER-selective ligands. The ER antagonist ICI 182 780 reversed the suppressive effects of 17beta-estradiol (E2), demonstrating that the protective effects of E2 on disease are dependent upon ER signaling. Treatment of SJL mice with the ERalpha-selective agonist proteolipid protein (PPT) prior to the induction of disease resulted in suppression of clinical symptoms of disease, whereas treatment with an ERbeta-selective agonist (WAY-202041) had no effect. Treatment of mice with PLP peptide 139-151 (PPT) was also associated with decreased immune responses associated with disease. Consistent with its lack of effect on disease, the ERbeta agonist had minimal effects on immune responses. The use of selective estrogen receptor modulators (SERMs) in this model was also explored, and we show that raloxifene and WAY-138923 were also effective in suppressing disease. These results demonstrate the beneficial effects of estrogen receptor ligands, in particular ERalpha-selective ligands, and may have implications in the development of therapeutic strategies for multiple sclerosis.     

Defective DNA mismatch repair in long-term (> or =3 years) survivors with pancreatic cancer.

BACKGROUND/AIMS: Defective DNA mismatch repair (MMR) in pancreatic cancer, reported in up to 13% of sporadic pancreatic cancers, may predict a good prognosis. To determine if long-term survival in pancreatic cancer could be attributed to defective DNA MMR, we ascertained its prevalence in 35 pancreatic cancer patients who survived > or =3 years after surgery. METHODS: We performed immunohistochemistry (IHC) for MMR proteins hMLH1, hMSH2, and hMSH6 in all 35 tumors and microsatellite instability (MSI) studies in 34/35 tumors using 10 microsatellite markers in paired normal and tumor DNA. Defective DNA MMR was defined as absence of protein expression on IHC and/or MSI in > or =30% of markers studied. RESULTS: On IHC, 3/35 (8.6%) tumors had defective DNA MMR. All 3 had absent expression of a DNA MMR protein (hMLH1 in 2 and hMSH2) and 2/3 also had MSI; the third could not be tested. Definitely 2, and probably all 3 patients had hereditary nonpolyposis colon cancer as determined by clinical and genetic profiles. CONCLUSION: Defective DNA MMR is uncommon in long-term survivors of pancreatic cancer and does not account for the survival benefit in those with sporadic pancreatic cancer.     

Paraflagellar rod protein-specific CD8+ cytotoxic T lymphocytes target Trypanosoma cruzi-infected host cells

Our previous studies show that in mice immunized with the paraflagellar rod (PFR) proteins of Trypanosoma cruzi protective immunity against this protozoan parasite requires MHC class I-restricted T cell function. To determine whether PFR-specific CD8+ T cell subsets are generated during T. cruzi infection, potential CTL targets in the PFR proteins were identified by scanning the amino acid sequences of the four PFR proteins for regions of 8-10 amino acids that conform to predicted MHC class I H-2b binding motifs. A subset of the peptide sequences identified were synthesized and tested as target antigen in 51Cr-release assays with effector cells from chronically infected T. cruzi mice. Short-term cytotoxic T lymphocyte (CTL) lines specific for two of the peptides, PFR-1(164-171) and PFR-3(123-130), showed high levels of lytic activity against peptide-pulsed target cells, secreted interferon (IFN)-gamma in response to parasite-infected target cells, and were found to be CD8+, CD4-, CD3+, TCRalphabeta+ cells of the Tc1 subset. Challenge of PFR immunized CD8-/- and perforin-deficient (PKO) mice confirmed that while CD8+ cells are required for survival of T. cruzi challenge infection, perforin activity is not required. Furthermore, while lytic activity of PFR-specific CD8+ T cell lines derived from PKO mice was severely impaired, the IFN-gamma levels secreted by CTLs from PKO mice were equivalent to that of normal mice, suggesting that the critical role played by CD8+ T cells in immunity to the parasite may be secretion of type 1 cytokines rather than lysis of parasite infected host cells.     

射频消融房室结改良消融前后心脏电生理变化的探讨

目的比较房室结双径路伴房室结内折返性心动过速(AVNRT)患者,射频消融(RFCA)慢径路改良术,消融前、后心脏各部分腔内电生理改变。方法在相同条件下,于消融前、后分别进行腔内电生理检查。记录消融前、后:希氏束电图(HIS),心房有效不应期(A—ERP),功能不应期(A—FRP),心室有效不应期(V—ERP),功能不应期(V—FRP),房室结前传有效不应期(AVN—ERP),前传文氏点(AVN—WKB),房室结逆传有效不应期(VAN—ERP),逆传文氏点(VAN—WKB),将消融前、后心脏各部分电生理参数进行配对,经SPSS统计分析软件进行T检验分析。结果消融前、后:HIS电图,A—ERP,A—FRP,V—ERP,V—FRP,AVN—ERP,及VAN—WKB均无显著差异(P>0.05)。AVN—WKB,VAN—ERP有显著差异(P<0.05)。讨论射频消融房结改良对房室结双径路AVNRT疗效肯定。在消融前、后(急性期)房室结前、逆传电生理均有一定改变。这与消融改变了房室结的部分结构,如大部分病列慢径路消失有关。不同消融部位对房室结传导电理改变产生不同的结果。没有证据表明消融后,45岁以上年龄组房室结传导改变大于45岁以下年龄组。男女不同性别组之间亦无差异。