Mortality risk factors and fulminant sub-phenotype in anaerobic bacteremia: a 10-year retrospective, multicenter, observational cohort study.

PURPOSE: During the last decade, the incidence of anaerobic bacteremia (AB) has been increasing. Patients with AB may develop complex underlying diseases, which can occasionally be accompanied by fatal or fulminant outcomes. However, the risk factors for AB-related mortality remain unclear. Herein, we sought to elucidate the risk factors for AB-related mortality. METHODS: In this multicenter, retrospective, observational study, we enrolled patients with culture-proven AB from six tertiary hospitals in Japan, between January 2012 and December 2021. Data on patient and infection characteristics, laboratory findings, treatment, and outcome were collected, and their associations with mortality were analyzed. RESULTS: A total of 520 participants were included. The 30-day mortality in the study cohort was 14.0% (73 patients), and malignant tumors were frequently observed comorbidities in 48% of the entire cohort. Multivariable logistic regression analysis showed a Charlson comorbidity score of > 6, serum creatinine level of > 1.17 mg/dL, and hypotension to be independent risk factors for 30-day mortality in AB (odds ratios [ORs] 2.12, 2.25, and 5.12, respectively; p < 0.05), whereas drainage significantly reduced this risk (OR, 0.28; p < 0.0001). Twelve patients (2.3% of the whole cohort and 16.4% of the deceased patients) presented with extremely rapid progression leading to fatal outcome, consistent with "fulminant AB." CONCLUSIONS: This study identified acute circulatory dysfunction and performance of drainage as independent predictive factors for 30-day AB-related mortality and revealed the existence of a fulminant AB sub-phenotype. Our findings could serve as a practical guide to predict the clinical outcomes of AB.

Lactobacillus pentosus strain b240 suppresses pneumonia induced by Streptococcus pneumoniae in mice.

AIMS: Oral administration of probiotics has been known to improve inflammatory responses against infectious diseases. Here, we describe the inhibitory effect of oral intake of heat-killed Lactobacillus pentosus strain b240 (b240) on pneumococcal pneumonia in a murine experimental model. METHOD AND RESULTS: The mice treated with oral b240 for 21 days before Streptococcus pneumoniae infection exhibited prolonged survival time and less body weight loss, compared with saline-treated control mice. Mild pneumonia with significantly reduced secretion of inflammatory cytokines/chemokines according to related mitogen-activated protein kinase signalling molecules (phosphorylated c-Jun N-terminal kinase) was found in b240-treated mice, whereas severe pneumonia with hypercytokinemia was evident in control mice. Prominent reduction in the number of pneumococci and elevated expression of Toll-like receptor 2 and 4 in the lung tissues was concomitantly noted in b240-treated mice. CONCLUSIONS: These findings indicate that b240 has inhibitory effects on pneumococcal pneumonia induced by Strep. pneumoniae infection and improves inflammatory tissue responses, resulting in reduced damages to the respiratory tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that oral administration of b240 might protect host animals from Strep. pneumoniae infection by augmentation of innate immune response.

Requirement of CD9 on the egg plasma membrane for fertilization.

CD9 is an integral membrane protein associated with integrins and other membrane proteins. Mice lacking CD9 were produced by homologous recombination. Both male and female CD9-/- mice were born healthy and grew normally. However, the litter size from CD9-/- females was less than 2% of that of the wild type. In vitro fertilization experiments indicated that the cause of this infertility was due to the failure of sperm-egg fusion. When sperm were injected into oocytes with assisted microfertilization techniques, however, the fertilized eggs developed to term. These results indicate that CD9 has a crucial role in sperm-egg fusion.

Efficacy of autologous fat injection laryngoplasty with an adenoviral vector expressing hepatocyte growth factor in a canine model.

OBJECTIVE: The effectiveness of autologous fat injection laryngoplasty may be reduced by resorption of injected fat tissue. The aim of the present study was to clarify the efficacy of fat injection laryngoplasty using autologous fat plus a replication-defective adenoviral vector expressing hepatocyte growth factor, regarding reduction of injected fat tissue resorption. MATERIAL AND METHODS: Four female beagle dogs were used in this study. After sedation, a direct laryngoscope was introduced to enable visualisation of the larynx. In each dog, harvested autologous fat plus an adenoviral vector expressing hepatocyte growth factor was injected into the right true vocal fold, and harvested fat plus an adenoviral vector expressing no gene was injected into the left true vocal fold. A total laryngectomy was performed one year after the intracordal fat injection. Coronal sections of the resected whole larynges were made and the following parameters assessed using light and electron microscopy: size of fat area; number of vasculoendothelial cells surrounding adipocytes; and shape of injected adipocytes in the vocal fold. RESULTS: The fat area was significantly larger and the number of vasculoendothelial cells surrounding adipocytes significantly greater in the intracordal fat injection containing adenoviral vector expressing hepatocyte growth factor, compared with the control intracordal fat injection containing adenoviral vector expressing no gene. When viewed under electron microscopy, the injected adipocytes were observed to have grafted better in the intracordal fat injection with hepatocyte growth factor adenoviral vector, compared with the control intracordal fat injection with adenoviral vector expressing no gene. CONCLUSIONS: Injection into the vocal fold of autologous fat containing an adenoviral vector expressing hepatocyte growth factor can reduce subsequent resorption of injected fat.

Two-dimensional gel electrophoresis analysis in simultaneous influenza pneumonia and bacterial infection in mice.

Severe pneumonia is found in simultaneous influenza pneumonia and bacterial infection, and suggests a relationship with immunological mechanisms. Here, we performed two-dimensional gel electrophoresis to detect immunological molecules related to the fulminant pneumonia caused by influenza virus and Streptococcus pneumoniae co-infection in mice. We found two spots that were expressed strongly in co-infected mouse lungs, compared with S. pneumoniae or influenza virus singly infected mouse lungs. The spots were analysed by mass spectrometry, and identified as alpha-1 anti-trypsin (A1AT), known as an anti-protease for neutrophil-derived proteolytic enzymes, and creatine kinase, which reflects a greater degree of lung damage and cell death. A1AT expression was increased significantly, and proteolytic enzymes from neutrophils, such as neutrophil elastase, myeloperoxidase and lysozyme, were also secreted abundantly in influenza virus and S. pneumoniae co-infected lungs compared with S. pneumoniae or influenza virus singly infected lungs. These data suggest that A1AT may play a central role as a molecule with broad anti-inflammatory properties, and regulation of the neutrophil-mediated severe lung inflammation is important in the pathogenesis of co-infection with influenza virus and bacteria.

Gabexate mesilate suppresses influenza pneumonia in mice through inhibition of cytokines.

Gabexate mesilate is a synthetic protease inhibitor that is effective for acute pancreatitis. The effect of gabexate mesilate in influenza pneumonia in mice was investigated by examining the changes in pulmonary inflammatory cytokines and chemokines. Pathological changes in the lungs of treated mice were extremely mild, compared with changes in infected, untreated mice. Intrapulmonary levels of interleukin-6 and macrophage inflammatory protein-2 decreased in treated mice compared with untreated mice, despite similar viral titres in the lungs. Survival terms for treated and untreated groups were similar. These data indicate that gabexate mesilate has beneficial effects on influenza pneumonia, which may be due to the modulation of inflammatory cytokine/chemokine responses.

Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis.

Immune and inflammatory systems are controlled by multiple cytokines, including ILs and INFs. These cytokines exert their biological functions through Janus tyrosine kinases and STAT transcription factors. One such cytokine, IL-6, has been proposed to contribute to the development of rheumatoid arthritis (RA). We found that STAT3 was strongly tyrosine phosphorylated in synovial tissue of RA patients, but not those with osteoarthritis. Blockade of the IL-6-gp130-JAK-STAT3-signaling pathway might therefore be beneficial in the treatment of RA. We show here that the mRNA for the endogenous cytokine signaling repressor CIS3/SOCS3 is abundantly expressed in RA patients. To determine whether CIS3 is effective in treating experimental arthritis, a recombinant adenovirus carrying the CIS3 cDNA was injected periarticularly into the ankle joints of mice with antigen-induced arthritis or collagen-induced arthritis (CIA). Periarticular injection of CIS3 adenovirus drastically reduced the severity of arthritis and joint swelling compared with control groups. CIS3 was more effective than a dominant-negative form of STAT3 in the CIA model. Thus, induction of CIS3 could represent a new approach for effective treatment of RA.

Combination suicide and cytokine gene therapy for hepatic metastases of colon carcinoma: sustained antitumor immunity prolongs animal survival.

The effectiveness of combination therapy using a suicide gene and cytokine genes for the treatment of metastatic colon carcinoma in the mouse liver was investigated. Pre-established hepatic tumors treated with a recombinant adenoviral vector containing the herpes simplex virus thymidine kinase gene(tk) exhibited substantial regression, although all treated animals suffered from subsequent relapses. Although cotreatment with a mouse interleukin 2 (mIL-2)-containing adenoviral vector induced an effective antitumor immune response, the immunity waned with time, and the treated animals eventually succumbed to hepatic tumor relapse or distant metastases. In this study, mouse granulocyte macrophage colony-stimulating factor (mGM-CSF) gene was tested for its ability to further enhance and prolong the antitumoral cellular immunity. A fraction of the animals treated with tk + mIL-2 + mGM-CSF developed long-term antitumor immunity and survived for more than 4 months without recurrence. This long-term antitumor immunity could be enhanced further by subsequent "vaccination" with mIL-2-expressing parental tumor cells. The results indicate that local expression of GM-CSF in the hepatic tumors and prolonged mIL-2 expression are necessary to generate persistent antitumor immunity that is essential for the prevention of tumor recurrence and long-term animal survival.

The role of interleukin-2 in combination adenovirus gene therapy for head and neck cancer.

Interleukin-2 (IL-2) gene therapy alone and in combination with the herpes thymidine kinase gene (tk) was used to evaluate immunological responses and antitumor effects in head and neck cancer. Established floor of mouth squamous cell carcinomas in C3H/HeJ mice were directly injected with recombinant adenoviral vectors carrying both therapeutic and control genes. One week after adenoviral gene transfer, only the animals treated with combination IL-2+tk or tk alone demonstrated significant tumor regression. Residual tumors were harvested for microscopic evaluation and immunohistochemistry staining, which revealed a predominance of CD8+ lymphocytes in the tumor beds of the animals treated with IL-2. To evaluate the systemic immune effects of IL-2, animals treated with single or combination gene therapy received a second site challenge with parental tumor cells or a heterologous but syngeneic sarcoma cell line. Mice treated with combination IL-2 and tk demonstrated a protective systemic immunity specific to the parental tumor cell line, whereas no systemic immune response was evident in mice receiving IL-2 alone. In a separate experiment, a range of concentrations of the adenovirus IL-2 vector were used to treat established tumors. Even with the maximal single-dose adenovirus concentration, IL-2 alone was ineffective as a single therapy. These results support the use of adenovirus-mediated gene transfer of IL-2 as an effective immunotherapy when used adjuvantly with the tk "suicide gene".

Efficacy of topical antibiotic administration on the inhibition of perioperative oral bacterial growth in oral cancer patients: a preliminary study.

Parenteral antibiotic prophylaxis is the current standard of therapy in clean-contaminated oral cancer surgery. Nevertheless, the incidence of surgical site infection (SSI) in oral oncological surgery is relatively high, especially in major surgery with reconstruction and tracheotomy. The aims of this study were to investigate the perioperative condition related to microorganisms in the oral cavity and to examine the efficacy of the topical administration of tetracycline in reducing the number of bacteria in the oropharyngeal fluid during intubation. The number of oral bacteria was measured during intubation in patients undergoing major oral cancer surgery. The efficacy of the topical administration of tetracycline or povidone iodine gel in reducing the bacteria was then investigated. Bacteria in the oropharyngeal fluid grew from 10(6)CFU/ml to 10(8)CFU/ml during the 3h after intubation (CFU, colony-forming units). When tetracycline was applied to the dorsum of the tongue, oral bacteria decreased immediately to 10(5)CFU/ml, and the number of bacteria in the oropharyngeal fluid was maintained below 10(7)CFU/ml for 7h. The concentration of tetracycline in the oropharyngeal fluid was extremely high for several hours after topical administration. The topical administration of tetracycline could reduce oral bacteria in patients undergoing clean-contaminated oral cancer surgery. This method is expected to be effective in the prevention of SSI.

Retrovirus-mediated in vivo gene transfer in the replicating liver using recombinant hepatocyte growth factor without liver injury or partial hepatectomy.

Retrovirus-mediated gene delivery into hepatocytes in vivo provides long-term gene expression, which is of great importance for treating most genetic and metabolic disorders. However, clinical application has not been realized because of the requirement for prior 70% partial hepatectomy or chemical (toxic) liver injury to initiate hepatocyte replication at the time of retroviral gene transduction. In this paper, we describe a novel gene delivery system that uses recombinant hepatocyte growth factor (rHGF) prior to retrovirus-mediated in vivo gene transfer in the liver without partial hepatectomy or liver injury. A single retroviral infusion through the portal vein following five systemic injections (via the tail vein) of 100 microg/kg rHGF resulted in a 10.4% 5-bromo-2'-deoxyuridine (BrdU) labeling index (BLI) and 0.14% retroviral gene transduction efficiency (RGTE) in hepatocytes, which were 6.3- and 12.9-fold higher than those of controls, respectively. Modest additional increases in BLI and RGTE (13.4% and 0.22%, respectively) were seen after five systemic injections of 500 microg/kg rHGF. The correlation between BLI and RGTE was statistically confirmed regardless of treatment. When rats received multiple retroviral infusions through a cannulated portal vein following five portal injections of 100 microg/kg rHGF, RGTE was dramatically increased (1.3%) and in some areas of the liver exceeded more than 10%. There was no evidence of liver injury in any animal. This approach has great potential for clinical application in terms of avoiding invasive procedures or liver injury.

Adenoviral-mediated herpes simplex virus-thymidine kinase gene transfer in vivo for treatment of experimental human melanoma.

To assess the efficacy of an in vivo adenoviral-mediated cytotoxic gene therapy, human melanomas were established in nude mice and transduced with herpes simplex virus-thymidine kinase (tk) followed by treatment with ganciclovir (GCV). In initial experiments, adenovirus (adv) containing the beta-galactosidase reporter gene was employed to determine melanoma cell infectivity in vitro. In comparison to murine melanoma cell lines B16 and K1735-M2, human A375-SM cells exhibited up to a 10-fold greater susceptibility to adenoviral transduction, similar to the degree of infectivity found for human epidermal HaCaT cells. In addition, human A375-SM melanoma cells exhibited a greater sensitivity in vitro to the cytotoxic effects of transduction with tk-adv and treatment with GCV, which was mediated by a strong bystander effect. In vivo, intratumoral injection of relatively large human melanomas (160 mm3) with 1.2 X 109 pfu of tk-adv, followed by intraperitoneal GCV treatment (60 mg/kg twice daily) over 4 days, typically resulted in a 50% reduction in melanoma growth rate compared to mock or untreated controls. Moreover, histometrical analysis employing a rigorous computerized imaging system revealed that the residual viable tumor area in the tk-adv/GCV-treated group was only one-fifth that of solvent controls. These data show that adv is a highly efficient in vivo gene delivery system to treat experimental human melanomas. In comparison to a previous murine melanoma study, human melanomas appeared to exhibit a greater sensitivity to this cytotoxic treatment in vivo, which may hold significant promise for development of effective gene therapy modalities to treat melanoma in humans.

Adenoviral-mediated suicide gene therapy for hepatic metastases of breast cancer.

Metastases of breast cancer are a major cause of treatment failure. To evaluate the therapeutic efficacy of suicide gene therapy in metastatic breast cancer, we used the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) administration to treat breast cancer, generated by an adenocarcinoma cell line MOD in syngeneic mice. The bystander effect of HSV-tk + GCV on tumor cell killing was illustrated by demonstrating complete regression of subcutaneous tumors consisting of 90% parental tumor cells and 10% HSV-tk transformed tumor cells. To establish a model of breast cancer metastases in the liver, tumors were generated by intra-hepatic implantation of MOD cells in syngeneic animals. Two weeks after tumor cell implantation, replication defective adenoviral vectors expressing HSV-tk (ADV.tk), or beta-galactosidease (ADV. beta-Gal) were injected intratumorally, followed by buffer or GCV administration. Treatment with ADV.tk + GCV resulted in significant regression of tumor (P < .001), as assessed by computerized morphometric analysis of residual tumor. This was reflected as a significant prolongation of survival in treated animals (P < .001). These results demonstrate that ADV-mediated suicide gene therapy in vivo can be incorporated in a comprehensive treatment strategy for liver metastases of breast cancer.

Combination therapy with suicide and cytokine genes for hepatic metastases of lung cancer.

Metastases of lung cancer are a major cause of treatment failure. To evaluate the therapeutic efficacy of gene therapy in metastatic lung cancer, we used adenoviral (ADV) mediated transfer of the herpes simplex virus thymidine kinase (HSV-tk) gene and the cytokine gene interleukin-2 (IL-2) to treat a murine model of metastatic lung cancer in the liver. Hepatic metastases were established by intrahepatic implantation of LL2 cells in syngeneic recipient mice. One week after tumor implantation, various replication defective ADV vectors were injected intratumorally. Treatment with a vector expressing the HSV-tk followed by ganciclovir administration with ADV.tk resulted in significant regression of tumor (p<0.01) as well as prolongation of survival (p<0.001). While a vector expressing mouse IL-2 ADV.IL-2 alone was ineffective, combination therapy with HSV-tk resulted in further tumor regression and improvement of animal survival (p<0.05). These results demonstrate that suicide and cytokine genes can be utilized in combination to treat metastatic lung cancer in vivo.

Influences of a dietary fatty acid composition on the emergence of glutathione S-transferase-P (GST-P) positive foci in the liver of carcinogen-treated rats.

The rats treated with a single i.p. injection of diethylnitrosoamine (DEN) and percial hepatectomy were fed for 11 weeks with a high fat diet mixed with 10% lard, eicosapentaenoic-acid-rich oil (EPA-oil) or arachidonic-acid-rich oil (AA-oil) and the emergence of glutathione S-transferase placental form (GST-P) in the liver was evaluated. There were no significant differences in the serum aminotransferase activities. The molar ratio of n-6 and n-3 fatty acid in the liver phospholipids was significantly low in the EPA-oil group compared with the other groups. In the EPA-oil group, the area percent and the unit area of GST-P positive foci were significantly smaller than the other groups. In the AA-oil group, no significant differences were recognized in the quantitative values for GST-P positive foci compared with the control and lard groups. In conclusion, a hepatic neoplasmic lesion induced by DEN was suppressed with EPA-rich fish oil, and arachidonic-acid-rich oil showed no effect of suppression or acceleration.

MK-801 reduces non-noxious stimulus-evoked Fos-like immunoreactivity in the spinal cord of rats with chronic constriction nerve injury.

We investigated the role of N-methyl-D-aspartate (NMDA) receptors on non-noxious stimulus-induced pain by examining the effect of MK-801, a non-competitive NMDA receptor antagonist, on Fos-like immunoreactivity (FLI) in the spinal dorsal horn by non-noxious stimulation to rats with chronic constriction injury (CCI) of the sciatic nerve. In CCI rats that did not receive the non-noxious stimulus, FLI was significantly increased in laminae V/VI of the dorsal horn at the 7th and 14th days after surgery relative to sham rats. When CCI rats received non-noxious stimuli, rubbing the plantar of the hind paw, FLI in laminae I/II at the 14th day was significantly increased relative to CCI rats that did not receive the stimulation. In sham rats, the same stimulus significantly decreased FLI in laminae III/IV and V/VI at the 7th and 14th day. When MK-801 was administered intraperitoneally prior to non-noxious stimulation in CCI rats at the 14th day after surgery, the stimulus-induced FLI in laminae I/II in CCI rats was significantly reduced. This study indicates that NMDA receptor is involved in upregulating FLI in response to non-noxious stimulation of CCI rats.

Adenoviral-mediated herpes simplex virus thymidine kinase gene transfer: regression of hepatic metastasis of pancreatic tumors.

Pancreatic cancer is the fifth leading cause of cancer death in the United States. Most patients have obvious metastases or locally advanced disease at the time of presentation. Surgical resection does not significantly change the clinical outcome. Combination chemotherapy induces a partial response but overall survival remains low. The aim of this study was to evaluate the feasibility of adenovirus-mediated suicide gene transduction as a therapeutic approach for pancreatic cancer. A cell line was established from a murine pancreatic ductal adenocarcinoma and intrahepatic tumors were generated by inoculation of pancreatic cancer cells into the left lateral liver lobe. Transduction efficiency was characterized in vitro and in vivo. Intrahepatic tumors were treated by intratumoral adenovirus injection in combination with intraperitoneal administration of ganciclovir. Adenovirus-mediated herpes simplex virus (HSV)-thymidine kinase (tk) gene expression followed by ganciclovir treatment was highly efficient in inhibiting pancreatic cancer cell proliferation in vitro. The proliferation of nontransduced cells was significantly reduced in the presence of HSV-tk expressing cells. Intrahepatic inoculation of pancreatic cancer cells leads to successful formation of solid adenocarcinomas in syngeneic recipients. Ad.RSV-tk injection of the tumor followed by intraperitoneal ganciclovir application caused highly significant tumor volume reduction and necrosis. These results indicate that transduction of the HSV-tk gene followed by ganciclovir is highly efficient for growth inhibition of hepatic metastases of pancreatic carcinoma.

Infantile cholestasis due to cytomegalovirus infection of the liver. A possible cause of paucity of interlobular bile ducts.

We report a neonatal case of hepatic cytomegalovirus (CMV) infection associated with paucity of interlobular bile ducts. The premature infant developed severe jaundice, and died of disseminated CMV infection. The diagnosis of CMV infection was based on detection of pathognomonic inclusions in the liver and on a high titer of CMV antibody in the serum. The hepatic histologic findings in this infant included severe cholestasis, complete absence of interlobular bile ducts in the majority of the portal tracts, and CMV inclusions in the duct epithelium. Interestingly, the CMV inclusions were localized in the bile duct, but not in the parenchyma. We postulate that the paucity of interlobular bile ducts may have been a sequela of CMV infection of the liver arising from destruction of the bile ducts, leading to severe cholestasis.

Xanthogranulomatous pyelonephritis, perinephric type--a case report.

A 46 year-old woman with perinephric type of xanthogranulomatous pyelonephritis is described. She had a fever and pain with a palpable mass in her right flank. The blood analysis revealed anemia, leucocytosis, gamma-globulinemia, but no hyperlipidemia. The urine analysis showed nothing abnormal, but enterobacter was present in the urine. An intravenous pyelogram demonstrated a right non-functioning kidney. The diagnosis of a perinephric abscess was made from the x-ray and ultrasonogram, and a right nephrectomy was performed. The resected kidney had a tumor-like lump covered with Gerota's fascia at the postero-lateral side of the kidney. The cut surface of the kidney revealed an area of hemorrhage, blood clotting, abscess and a brownish yellow area in the perinephric fat tissue. The calyx and pelvis were normal. Histologically, the brownish yellow area was a granuloma with foam cell infiltration. The foam cells contained lipids. The renal parenchyma showed a non-specific chronic pyelonephritis.

Ultrastructural studies of the bile duct in alcoholic liver disease.

The fine structural characteristics of the bile duct in patients with alcoholic disease are described. Dark cell metamorphosis, edematous microvilli, and increased number of pinocytotic vesicles on the basal wall surface of the duct epithelium were observed. These alterations may be interpreted as evidence of disordered water metabolism, probably reflecting secretion and reabsorption hyperfunction in the duct epithelium. In addition, widened intercellular spaces in the basal half of the epithelium suggested retention of fluid following reverse pinocytosis along the lateral cell surface. Although no alterations of the duct epithelium distinct from those in patients with other liver diseases were apparent in patients with alcoholic liver disease, the basement membrane of the bile duct exhibited unusual duplication with multiple layers and occasional loop-formation in lacunae on the basal surface.