Catheter-associated bloodstream infection in patients with cancer: comparison of left- and right-sided insertions.

BACKGROUND: There is limited research on the relationship between side of insertion of central venous catheter (CVAD) and bloodstream infection risk in patients with cancer. AIM: To conduct an exploratory analysis of data from a randomized control trial (RCT) and data from a prospective cohort study to compare infection rates for right- and left-sided insertions. METHODS: The study populations were patients aged >14 years with cancer from two tertiary hospitals in Brisbane, Australia. The primary endpoint was catheter-associated bloodstream infection (CABSI) adjudicated by blinded assessors. For the RCT, randomized intention-to-treat comparisons were conducted between left- and right-side allocated insertion for early (≤14 days) and late (>14 days) infection using Cox proportional hazards regression. The RCT data were also combined with cohort study data collected from one of the hospitals prior to the RCT and non-randomized comparisons conducted between left- and right-sided insertions. FINDINGS: In 634 randomly allocated CVADs there were 141 CABSIs. Analysis showed strong evidence of right-side allocated insertions having an increased risk of early infection by 2.5 times (95% confidence interval (CI): 1.3-4.7); however, there was no evidence of increased risk for late infection (hazard ratio: 1.06; 95% CI: 0.71-1.59). Results from analysis of the RCT and cohort study data combined (2786 CVADs and 385 CABSIs) were similar. CONCLUSION: There appears to be an increased risk of CABSI in patients with cancer for CVAD inserted into the right-side for around two weeks after line insertion. The mechanism underpinning the increased risk is unknown.

Kidney biopsy practice amongst Australasian nephrologists.

BACKGROUND: Percutaneous kidney biopsy is the gold standard investigation for the diagnosis of kidney diseases. The associated risks of the procedure depend on the skill and experience of the proceduralist as well as the characteristics of the patient. The Kidney Health Australia - Caring for Australasians with Renal Impairment (KHA-CARI) guidelines on kidney biopsies, published in 2019, are the only published national kidney biopsy guidelines. As such, this study surveys current kidney biopsy practices in Australasia and examines how they align with the Australian guidelines, as well as international biopsy practice. METHODS: A cross-sectional, multiple-choice questionnaire was developed examining precautions prior to kidney biopsy; rationalisation of medications prior to kidney biopsy; technical aspects of kidney biopsy; complications of kidney biopsy; and indications for kidney biopsy. This was distributed to all members of the Australian and New Zealand Society of Nephrology (ANZSN). RESULTS: The response rate for this survey is approximately 21.4 % (182/850). Respondents found agreement (> 75.0 %) in only six out of the twelve questions (50.0 %) which assessed their practice against the KHA-CARI guidelines. CONCLUSIONS: This is the first study of its kind where kidney biopsy practices are examined against a clinical guideline. Furthermore, responses showed that practices were incongruent with guidelines and that there was a lack of consensus on many issues.

Effect of suberoylanilide hydroxamic acid on peripheral blood mononuclear cell cytotoxicity towards tumor cells in canines.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor (HDACi) that suppresses the growth of tumor cells in humans and canines. SAHA reportedly enhances the antitumor activity of human peripheral blood mononuclear cell (PBMC). However, it is unclear whether a similar effect is exerted in canines. The present study focused on the effect of SAHA on the cytotoxicity of IL-2 activated PBMC in three tumor cell lines (CTAC, CIPm, and MCM-N1). The mRNA expression of a ligand for the NKG2D receptor was upregulated in SAHA-treated cell lines. Moreover, the SAHA-treated cell lines, except MCM-N1 demonstrated a significantly higher PBMC cytotoxicity compared to the untreated cell lines. Therefore, the NKG2DL upregulation likely enhanced the interaction of NKG2D-NKG2DL, leading to enhanced cytotoxicity of PBMC. It was also revealed that activated PBMC treated with SAHA significantly attenuated their cytotoxicity toward all the cell lines. Although the NKG2D, NKp46, NKp44, and NKp30 receptors, involved in PBMC cytotoxicity, were presumed to be downregulated, there was no significant reduction in the mRNA expression of these receptors. This study revealed that SAHA not only sensitizes the canine tumor cells to cytotoxicity due to PBMC activation, but also suppresses the cytotoxicity of PBMC themselves. Therefore, our results highlight the necessity of avoiding this inhibitory action to enhance the antitumor effect of SAHA in canines.

The incidence of postoperative nausea and vomiting after caesarean section in patients with hyperemesis gravidarum: a retrospective cohort study.

BACKGROUND: Postoperative nausea and vomiting is one of the most common anaesthetic complications of caesarean section. This study examined the association between hyperemesis gravidarum during pregnancy and nausea and vomiting after caesarean section. METHODS: A single-centre, retrospective cohort study, using electronic databases of patients with and without hyperemesis gravidarum, undergoing caesarean section from 2015 to 2019. The incidence and severity of postoperative nausea and vomiting were established by a review of the documentation of administration of postoperative anti-emetics within the 24-h period after surgery, and examined using univariable, multivariable binary and ordered logistic regression models. RESULTS: Data were compared for 76 patients with hyperemesis gravidarum and 315 patients without the condition. The incidence of postoperative nausea and vomiting in the hyperemesis group versus the non-hyperemesis group was 43.4% vs 29.6%, respectively. The odds of experiencing postoperative nausea and vomiting was 1.95 times higher in women with hyperemesis gravidarum than in those without (aOR 1.95, 95% CI 1.13 to 3.36, P=0.016). The odds of having more severe postoperative nausea and vomiting were greater in the hyperemesis gravidarum group (aOR 1.91, 95% CI 1.14 to 3.20, P=0.014). CONCLUSION: Patients with hyperemesis gravidarum are more likely to develop nausea and vomiting after caesarean section, and this is likely to be of greater severity than in those without the condition. This finding should assist the effective provision of intra-operative and postoperative anti-emetics for patients with hyperemesis gravidarum undergoing caesarean section.

Catheter-associated bloodstream infections in adults with cancer: a prospective randomized controlled trial.

BACKGROUND: A common complication of central venous access devices (CVADs) is catheter-associated bloodstream infection (CABSI). We previously demonstrated that insertion of CVADs on the right side was associated with increased risk of CABSI, and hypothesized that this related to the predominance of right-handedness in the patient population, resulting in greater movement and bacterial contamination. AIM: To perform a prospective randomized, controlled, non-blinded study to determine whether the side of CVAD insertion influenced the incidence of CABSI. METHODS: Adult cancer patients were randomly allocated to either dominant or non-dominant side CVAD insertion. The primary endpoint of the study was the number of line-days until CABSI, determined in a blinded fashion by two assessors. FINDINGS: In all, 640 CVADs were randomized to dominant (N = 322) or non-dominant (N = 318) side of insertion, 60% had haematological malignancies, and 40% solid tumours. CVADs were a peripherally inserted central catheter line (67%), tunnelled CVAD (23%), and non-tunnelled CVAD (10%). Twenty-two percent of CVADs were complicated by CABSI. The rate of CABSI per 1000 line-days was 3.49 vs 3.66 in the non-dominant vs dominant group (hazard ratio (HR): 0.91; 95% confidence interval (CI): 0.65-1.28). By multivariable analysis, the rate of CABSI was increased by: use of tunnelled CVADs compared to peripherally inserted central venous catheter lines (HR: 2.05; 95% CI: 1.45-2.91); having a haematological malignancy compared to non-gastrointestinal solid tumours (5.55; 2.47-12.5); but not dominant compared to non-dominant side of CVAD (0.97; 0.69-1.36). CONCLUSION: CABSI in adult patients with cancer was not impacted by whether CVAD insertion was on the dominant or non-dominant side.

Alteration in faecal bile acids, gut microbial composition and diversity after laparoscopic sleeve gastrectomy.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is a well established treatment for severe obesity and type 2 diabetes. Although the gut microbiota is linked to the efficacy of LSG, the underlying mechanisms remain elusive. The effect of LSG for morbid obesity on the gut microbiota and bile acids was assessed here. METHODS: Severely obese subjects who were candidates for LSG were included and followed until 6 months after surgery. The composition and abundance of the microbiota and bile acids in faeces were assessed by 16S ribosomal RNA sequencing, quantitative PCR and liquid chromatography-mass spectrometry. RESULTS: In total, 28 patients with a mean(s.d.) BMI of 44·2(6·6) kg/m2 were enrolled. These patients had achieved excess weight loss of 53·2(19·0) per cent and showed improvement in metabolic diseases by 6 months after LSG, accompanied by an alteration in the faecal microbial community. The increase in α-diversity and abundance of specific taxa, such as Rikenellaceae and Christensenellaceae, was strongly associated with reduced faecal bile acid levels. These changes had a significant positive association with excess weight loss and metabolic alterations. However, the total number of faecal bacteria was lower in patients before (mean(s.d.) 10·26(0·36) log10 cells per g faeces) and after (10·39(0·29) log10 cells per g faeces) operation than in healthy subjects (10·83(0·27) log10 cells per g faeces). CONCLUSION: LSG is associated with a reduction in faecal bile acids and greater abundance of specific bacterial taxa and α-diversity that may contribute to the metabolic changes.

ANTECEDENTES: La gastrectomía vertical laparoscópica (laparoscopic sleeve gastrectomy, LSG) es un tratamiento bien establecido para la obesidad grave y la diabetes tipo 2. Aunque la microbiota intestinal se ha vinculado con la eficacia de LSG, los mecanismos subyacentes siguen siendo poco conocidos. En este estudio se evaluó el efecto de LSG en la obesidad mórbida sobre la microbiota del intestino y de los ácidos biliares (bile acids, BA). MÉTODOS: Tras la aprobación del Comité ético y la obtención del consentimiento informado, los sujetos con obesidad grave que eran candidatos para LSG fueron incluidos en el estudio y seguidos durante 6 meses después de la operación. Se evaluaron la composición y abundancia de la microbiota y BA en las heces mediante secuenciación del gen 16S rRNA, PCR cuantitativa y cromatografía líquida-espectrometría de masas. RESULTADOS: En total, 28 pacientes con una mediana (rango) del IMC de 43,9 kg/m2 (35,0-61,9) fueron reclutados y a los 6 meses tras una LSG, consiguieron una pérdida del exceso de peso de 47,3% (20,7-95,1) y mejoría de las enfermedades metabólicas acompañada de una alteración en la comunidad microbiana fecal. El aumento en la diversidad α y abundancia de especies taxonómicas específicas como Rikenellaceae y Christensenellaceae, se asociaba fuertemente con niveles fecales reducidos de BA. Estos cambios se asociaban de manera positiva y significativa con la pérdida del exceso de peso y las alteraciones metabólicas. Sin embargo, el número total de bacterias fecales en los pacientes fue inferior al de los sujetos sanos (10,84 log10 células/g heces (9,46-11,35)) antes de la operación (10,26 log10 células/g heces (9,44-10,91)) y después de la misma (10,42 log10 células/g heces (9,57-10,96)). CONCLUSIÓN: LSG se asoció con menos BA fecal y mayor abundancia de especies bacterianas específicas y diversidad α lo que puede contribuir a los cambios metabólicos.

Cytotoxicity effect of trastuzumab on canine peripheral blood mononuclear cells.

BACKGROUND: Trastuzumab is an antibody drug used to treat human epidermal growth factor receptor 2 (HER2) overexpressing human metastatic breast cancer. Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism of cytotoxicity of the drug. However, its ability to induce an ADCC response in canine peripheral blood mononuclear cells (PBMCs) is not well established. AIMS: We aimed to evaluate the ability of trastuzumab in enhancing the cytotoxicity of PBMCs against canine tumor cells. METHODS: We used canine tumor cell lines isolated from metastatic mammary gland tumors (CHMm and CIPm) and thyroid adenocarcinoma (CTAC). The binding of trastuzumab to the cells was confirmed using flow cytometry analysis. Peripheral blood mononuclear cells obtained from healthy beagles and lymphokine-activated killer (LAK) cells, generated by interleukin-2 (IL-2) stimulation of PBMCs, were used as effector cells. Standard lactate dehydrogenase (LDH) release assay was used to measure the cytotoxicity of the LAK cells against tumor cell lines in the presence of trastuzumab. RESULTS: Trastuzumab enhanced the cytotoxicity of PBMCs against CHMm. Moreover, LAK cells killed CHMm synergistically in the presence of trastuzumab. However, the presence of trastuzumab did not produce such a synergistic effect when LAK cells acted against CIPm and CTAC. CONCLUSION: We confirmed the ability of trastuzumab to induce an ADCC response in canine PBMCs and determined its synergistic effect with LAK cells. Although the in vitro system in the present study did not show the induction of trastuzumab-mediated ADCC response against all canine tumor cell lines, the results of this study indicate the potential antitumor activity of trastuzumab in canines.

Influence of Trendelenburg position and pneumoperitoneum treatment on gastroesophageal reflux in dogs.

Gastroesophageal reflux is a latent factor that may cause esophagitis, esophageal stenosis, and aspiration pneumonia through the regurgitation of the gastric fluid contents. For laparoscopic surgery, posture-changing and pneumoperitoneum operations are conducted to develop the visual field. However, few studies have examined the influence of these operations on gastroesophageal reflux. In this experiment using 10 Beagles, 10 mL of contrast medium was administered into the stomach, and the dogs were placed in the Trendelenburg position with 10-degree tilting. Pneumoperitoneum treatment with carbon dioxide was performed, with an intraperitoneal pressure of 10 mmHg. The presence or absence of gastroesophageal reflux was evaluated using computed tomography (CT). In horizontal and Trendelenburg positions, there was no reflux of Contrast medium. However, reflux was observed in the Trendelenburg position under pneumoperitoneum (p<0.05). These results suggest that the risk of gastroesophageal reflux increases during laparoscopic surgery in the Trendelenburg position with 10-degree tilting under an intraperitoneal pressure of 10 mmHg.

Impact of ex vivo administration of mesenchymal stem cells on the function of kidney grafts from cardiac death donors in rat.

BACKGROUND: Mesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia-reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue-derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD). METHODS: Using male Lewis rats (8-10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase-LacZ) transgenic Lewis rats were injected either systemically (1.0 × 10(6) cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 µL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, which were then preserved (1 hour; 22°C) before being transplanted into bilaterally nephrectomized recipient rats (n = 8). Serum was collected to assess the therapeutic effects of AT-MSC administration, and the recipients of rats surviving to Day 14 were separately evaluated histopathologically. Follow-up was by in vivo imaging and histological LacZ staining, and tumor formation was evaluated in MSC-injected rats at 3 months. RESULTS: Systemic injection of MSC did not improve recipient survival. In vivo imaging showed MSCs trapped in the lung that later became undetectable. Ex vivo injection of MSCs did show a benefit without adverse effects. At Day 14 after RTx, 75% of the rats in the AT-MSC-injected group (MSC[+]) had survived, whereas 50% of the rats in the AT-MSC-non-injected group (MSC[-]) had died. Renal function in the MSC(+) group was improved compared with that in the MSC(-) group at Day 4. LacZ staining revealed AT-MSCs attached to the renal tubules at 24 hours after RTx that later became undetectable. Histopathologic examination showed little difference in fibrosis between the groups at Day 14. No teratomas or other abnormalities were seen at 3 months.

Scanning electron microscopic study for pore formation of the greater omentum of cats.

The greater omentum of the cats is said to have a lace-like structure. However, there are only a few descriptions on whether pores exist, and there are not many morphological studies on this meshwork. In this study, the greater omentum of the cats was observed at each age of development using a scanning electron microscope. The greater omentum of the cats immediately after birth was found to be continuous, and no pores were observed. Also, development of microvilli was observed in the mesothelial cells on the surface of the greater omentum. In young cats at 3 months of age, small pores were sporadically observed, and at the ages of 6-12 months, there were more and larger pores. It was estimated that the pores on the greater omentum are formed in the process of moving from the movement of organs, such as the stomach, intestines and diaphragm, and the presence of these pores enables the passage of ascites between the omental bursa, the greater omentum and the serosal cavity of the wall without flowing through the omental foramen.

Phase II trial of preoperative S-1 plus cisplatin followed by surgery for initially unresectable locally advanced gastric cancer.

BACKGROUND: The aim of this study was to evaluate the efficacy and feasibility of preoperative chemotherapy with S-1 plus cisplatin in patients with initially unresectable locally advanced gastric cancer. METHODS: We enrolled patients with initially unresectable locally advanced gastric cancer because of severe lymph node metastases or invasion of adjacent structures. Preoperative chemotherapy consisted of S-1 at 80 mg/m(2) divided in two daily doses for 21 days and cisplatin at 60 mg/m(2) intravenously on day 8, repeated every 35 days. If a tumor decreased in size, patients received 1 or 2 more courses. Surgery involved radical resection with D2 lymphadenectomy. RESULTS: Between December 2000 and December 2007, 27 patients were enrolled on the study. No CR was obtained, but PR was seen in 17 cases, and the response rate was 63.0%. Thirteen patients (48.1%) had R0 resections. There were no treatment related deaths. The median overall survival time (MST) and the 3-year overall survival (OS) of all patients were 31.4 months and 31.0%, respectively. Among the 13 patients who underwent curative resection, the median disease-free survival (DFS) and the 3-year DFS were 17.4 months and 23.1%, respectively. The MST and the 3-year OS were 50.1 months and 53.8%, respectively. The most common site of initial recurrence after the R0 resection was the para-aortic lymph nodes. CONCLUSIONS: Preoperative S-1 plus cisplatin can be safely delivered to patients undergoing radical gastrectomy. This regimen is promising as neoadjuvant chemotherapy for resectable gastric cancer. For initially unresectable locally advanced gastric cancer, new trials using more effective regimens along with extended lymph node dissection are necessary.

Characterization of human-type monoclonal antibodies against reduced form of hemin binding protein 35 from Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: The gram-negative anaerobe Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We previously identified a 35 kDa surface protein (hemin binding protein 35; HBP35) from P. gingivalis that exhibited coaggregation activity, while additional analysis suggested that this protein possessed an ability to bind heme molecules. For development of passive immunotherapy for periodontal diseases, human-type monoclonal antibodies have been prepared using HBP35 as an antigen in TransChromo mice. In the present study, we focused on a single antibody, TCmAb-h13, which is known to inhibit heme binding to recombinant HBP35. The aim of our investigation was to clarify the redox-related function of HBP35 and consider the benefits of human-type monoclonal antibodies. MATERIAL AND METHODS: To examine the antigen recognition capability of TCmAbs with immunoblotting and Biacore techniques, we used the native form as well as several Cys-to-Ser variants of recombinant HBP35. RESULTS: We found that the redox state of recombinant HBP35 was dependent on two Cys residues, (48) C and (51) C, in the thioredoxin active center (WCGxCx). Furthermore, TCmAb-h13 recognized the reduced forms of recombinant HBP35, indicating its inhibitory effect on P. gingivalis growth. CONCLUSION: Hemin binding protein 35 appears to be an important molecule involved in recognition of the redox state of environmental conditions. In addition, TCmAb-h13 had an inhibitory effect on heme binding to recombinant HBP35, thereby interfering with P. gingivalis growth.

Phase I trial of combination chemotherapy with docetaxel, cisplatin and S-1 (TPS) in patients with locally advanced or recurrent/metastatic head and neck cancer.

BACKGROUND: we investigated the maximum tolerated dose (MTD) of combination therapy with docetaxel, cisplatin, and S-1 (TPS) in patients with locally advanced or recurrent/metastatic head and neck cancer (HNC). PATIENTS AND METHODS: treatment consisted of docetaxel (Taxotere) at doses of 50, 60, and 70 mg/m(2); cisplatin at 70 mg·m(2)/day on day 1; and S-1 twice daily on days 1-14 at doses of 40, 60, and 80 mg·m(2)/day, repeated every 3 or 4 weeks. RESULTS: forty patients were enrolled. MTD was not reached until level 4. Subjects at expanded dose were limited to patients with locally advanced disease. Two dose-limiting toxic effects (DLTs) were observed at dose level 5 (TPS: 70/70/80 mg·m(2)/day, every 3 weeks), namely one grade 3 infection and one grade 3 hyperbilirubinemia, establishing this as the MTD. Of 12 patients treated at dose level 6 (TPS: 70/70/60 mg·m(2)/day, every 3 weeks), 2 DLTs were seen. Six achieved a complete response and 22 a partial response, giving a response rate of 70%. CONCLUSIONS: TPS was well tolerated. The recommended phase II dose as induction chemotherapy for locally advanced HNC was determined as 70/70/60 mg·m(2)/day every 3 weeks. Antitumor activity was highly promising and warrants further investigation.

Semi-allogeneic dendritic cells injected via the intratumoural injection route show efficient antitumour effects in cooperation with host-derived professional antigen-presenting cells.

Dendritic cells (DC)-based immunotherapy is a potent anticancer modality. In DC-based immunotherapy, allogeneic DC may be an alternative source, but the usefulness of allogeneic DC in DC-based immunotherapy is still controversial. When used for immunotherapy, three factors may affect the efficiency of an allogeneic DC-driven antitumour response: (1) survival time, which is affected by T-cell alloresponses; (2) major histocompatibility complex incompatibility with the host cells in the context of antigen presentation; and (3) the role of host-derived professional antigen-presenting cells (pAPC). In addition, it is unclear which injection route is preferable when using allogeneic DC. In this study, we demonstrate that semi-allogeneic DC, which share half of the genes of the recipient, are more effective when used via the intratumoural (i.t.) injection route, rather than the subcutaneous (s.c.) injection route, for the induction of efficient antitumour effects and the generation of a significant tumour-specific CD8(+) T-cell response. The i.t. route has the advantage of not requiring ex vivo pulsation with tumour lysates or tumour antigens, because the i.t.-injected DC can engulf tumour antigens in situ. Allogeneic bone marrow transplantation (BMT) models, which permit us to separately assess the three factors described previously, show that while all three factors are important for efficient antitumour effects, the control of the alloresponse to injected DC is the most crucial for host-derived pAPC to function well when DC are administered intratumourally. This information may be useful for DC-based cancer immunotherapy under circumstances that do not allow for the use of autologous DC.

Effects of lipopolysaccharide on the induction of mixed chimerism in cyclophosphamide-induced tolerance.

Cyclophosphamide (CP)-induced tolerance is a mixed chimerism-based tolerance and is one of the strategies used to induce transplant tolerance. Toll-like receptor (TLR) agonists are reportedly able to abrogate the induction of tolerance by activating alloreactive T cells, or by inhibiting Treg cells. However, little is known about the effect of the immune response mediated by TLR on mixed chimerism-based tolerance protocols. In this study, we evaluated the influence of lipopolysaccharide (LPS), which is best known as an TLR4 agonist, on CP-induced tolerance. BALB/c (H-2(d)) mice received a conditioning regimen consisting of 10(8) donor DBA/2 (H-2(d)) spleen cells (SC) on day 0 and 200 mg/kg CP on day 2. A single dose of 20 microg LPS was injected on day -2, 0, 7, or 35. Our results showed that LPS infusion at any time point resulted in chronic rejection of donor skin grafts and the abrogation of mixed chimerism in 33-60% of recipients. We found a correlation between skin graft acceptance and higher levels of mixed chimerism. Flow cytometric analysis revealed that donor-reactive T cells were permanently eliminated, regardless of LPS infusion. In conclusion, LPS-infusion had little influence on the immune response of donor-reactive T cells, but had a significant effect on the induction and maintenance of mixed chimerism in CP-induced tolerance.

Impact of deletion of envelope-related genes of recombinant Sendai viruses on immune responses following pulmonary gene transfer of neonatal mice.

We demonstrated previously that the additive-type recombinant Sendai virus (rSeV) is highly efficient for use in pulmonary gene transfer; however, rSeV exhibits inflammatory responses. To overcome this problem, we tested newly developed non-transmissible constructs, namely, temperature-sensitive F-deleted vector, rSeV/dF (ts-rSeV/dF) and a rSeV with all the envelope-related genes deleted (rSeV/dFdMdHN), for pulmonary gene transfer in neonatal mice, by assessing their toxicity and immune responses. The gene expression in the lungs of neonatal ICR mice peaked on day 2, then gradually decreased until almost disappearing at 14 days after infection in all constructs. Loss of body weight and mortality rate, however, were dramatically improved in mice treated with SeV/dFdMdHN (mortality=0%, n=41) and ts-rSeV/dF (24.2%, n=33) compared with additive rSeV (70.7%, n=58). Although the deletion of envelope-related genes of SeV had a small impact on the production of antibody and cytotoxic T-lymphocyte activity in both adults and neonates, a dramatic reduction was found in the events related to innate responses, including the production of proinflammatory cytokines, particularly in the case of neonates. These results indicate that pulmonary gene transfer using SeV/dFdMdHN warrants further investigation for its possible use in developing safer therapeutics for neonatal lung diseases, including cystic fibrosis.

Application of chimerism-based drug-induced tolerance to rat into mouse xenotransplantation.

The current critical shortage of human donor organs has stimulated the feasibility of the xenogenic transplantation, such as swine to primate. We have previously reported the induction of donor-specific tolerance in MHC-disparated recipient mice by using our cyclophosphamide (CP)-induced tolerance conditioning. In this study, we examined the efficacy of our CP-induced tolerance conditioning in xenogenic transplantation model. F344 rats and B10 mice were used as donors and recipients. Recipient mice were treated with donor spleen cells, CP, Busulfan and bone marrow cells, with or without prior NK-cell depletion. Donor mixed chimerism, and the presence of donor reactive T-cell population were analysed by flow cytometry. The survival of the donor skin grafts were observed after the conditioning. Donor mixed chimerism was temporary induced but terminated at 10 weeks after treatments. Donor-specific prolongation of the skin graft survival was observed after the treatments, however, grafts were rejected in the long term. NK-cell depletion, prior to the treatments, did not affect the levels of the mixed chimerism or graft prolongation. The donor-reactive recipient T-cell population was remained the same level as the untreated mice, suggesting the failure of the induction of the central T-cell tolerance. Thus, partial efficacy of our CP-induced tolerance treatments in the rat to mice xenotransplantation was observed. Our results suggested that the additional treatments were required to establish the stable xenogenic tolerance.

Recombinant Sendai virus vectors for activated T lymphocytes.

T-lymphocyte-directed gene therapy has potential as a treatment of subjects with immunological disorders. One current limitation of this therapeutic strategy is low gene transfer efficiency, even when complex procedures are used. We report herein that a recombinant Sendai virus vector (SeV) was able to overcome this issue. Using jellyfish enhanced green fluorescent protein gene (EGFP), we found that SeV was able to transduce and express a foreign gene specifically and efficiently in activated murine and human T cells, but not in naive T cells, without centrifugation or reagents including polybrene and protamine sulfate; the present findings were in clear contrast to those demonstrated with the use of retroviruses. The transduction was selective in antigen-activated T cells, while antigen-irrelevant T cells were not transduced, even under bystander activation from specific T-cell responses by antigens ex vivo. Receptor saturation studies suggested a possible mechanism of activated T-cell-specific gene transfer, ie, SeV might attach to naive T cells but might be unable to enter their cytoplasm. We therefore propose that the SeV vector system may prove to be a potentially important alternative in the area of T-cell-directed gene therapy used in the clinical setting.

Recombinant Sendai virus provides a highly efficient gene transfer into human cord blood-derived hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are a promising target for gene therapy, however, the low efficiencies of gene transfer using currently available vectors face practical limitations. We have recently developed a novel and efficient gene transfer agent, namely recombinant Sendai virus (SeV), and we have here characterized SeV-mediated gene transfer to human cord blood (CB) HSCs and primitive progenitor cells (PPC) using the jelly fish green fluorescent protein (GFP) gene. Even at a relatively low titer (10 multiplicity of infections), SeV achieved highly efficient GFP expression in CB CD34(+) cells (85.5+/-5.8%), as well as more immature CB progenitor cells, CD34(+)AC133(+) (88.2+/-3.7%) and CD34(+)CD38(-) (84.6+/-5.7%) cells, without cytokines prestimulation, that was a clear contrast to the features of gene transfer using retroviruses. SeV-mediated gene transfer was not seriously affected by the cell cycle status. In vitro cell differentiation studies revealed that gene transfer occurred in progenitor cells of all lineages (GM-CFU, 73.0+/-11.1%; BFU-E, 24.7+/-4.0%; Mix-CFU, 59+/-4.0%; and total, 50.0+/-7.0%). These findings show that SeV could prove to be a promising vector for efficient gene transfer to CB HSCs, while preserving their ability to reconstitute the entire hematopoietic series.

Mouse Tdho abnormality results from double point mutations of the emopamil binding protein gene (Ebp).

Mouse Tdho (Tattered-Hokkaido) was described as being allelic with Td in our previous study. Both allelic genes, which are located at the same position on the centromere of the X Chromosome (Chr), generate similar phenotypes such as male embryonic lethality, and in heterozygous females, hyperkeratotic skin, skeletal abnormalities, and growth retardation. The emopamil binding protein gene (Ebp) emerged as a candidate for mouse Tdho mutation, since the Td gene was recently determined to result from a point mutation of Ebp. In this study, Ebp cDNA of Tdho was demonstrated to possess double point mutations that cause two amino acid changes from Leu to Pro at position 132 and from Ser to Cys at 133 in EBP protein. EBP participates in cholesterol biosynthesis, and cholest-8(9)-en-3beta-ol was found to be increased in the plasma of Tdho adult females but not in that of normal mice. From these results, a loss of function was expected for the EBP protein encoded by Tdho. Both the phenotypes and genes responsible for Tdho as well as Td are quite similar to those of human X-linked chondrodysplasia punctata (CDPX2).