KRAS mutation leads to decreased expression of regulator of calcineurin 2, resulting in tumor proliferation in colorectal cancer.

KRAS mutations occur in 30-40% of all cases of human colorectal cancer (CRC). However, to date, specific therapeutic agents against KRAS-mutated CRC have not been developed. We previously described the generation of mouse models of colon cancer with and without Kras mutations (CDX2P-G22Cre;Apc(flox/flox); LSL-Kras(G12D) and CDX2P-G22Cre;Apc(flox/flox) mice, respectively). Here, the two mouse models were compared to identify candidate genes, which may represent novel therapeutic targets or predictive biomarkers. Differentially expressed genes in tumors from the two mouse models were identified using microarray analysis, and their expression was compared by quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemical analyses in mouse tumors and surgical specimens of human CRC, with or without KRAS mutations, respectively. Furthermore, the functions of candidate genes were studied using human CRC cell lines. Microarray analysis of 34 000 transcripts resulted in the identification of 19 candidate genes. qRT-PCR analysis data showed that four of these candidate genes (Clps, Irx5, Bex1 and Rcan2) exhibited decreased expression in the Kras-mutated mouse model. The expression of the regulator of calcineurin 2 (RCAN2) was also observed to be lower in KRAS-mutated human CRC. Moreover, inhibitory function for cancer cell proliferation dependent on calcineurin was indicated with overexpression and short hairpin RNA knockdown of RCAN2 in human CRC cell lines. KRAS mutations in CRC lead to a decrease in RCAN2 expression, resulting in tumor proliferation due to derepression of calcineurin-nuclear factor of activated T cells (NFAT) signaling. Our findings suggest that calcineurin-NFAT signal may represent a novel molecular target for the treatment of KRAS-mutated CRC.

Laparoscopic Hartmann's procedure for fecal peritonitis resulting from perforation of the left-sided colon in elderly and severely ill patients.

BACKGROUND: Traditional treatment for fecal peritonitis resulting from perforation of the left-sided colon has been performed using Hartmann's procedure to reduce the high mortality caused by anastomotic leakage. However, the morbidity rates associated with abdominal incision (due in great part to wound infection, and dehiscence of abdominal fascia) are high. Therefore, we propose using laparoscopic Hartmann's procedure with abdominal incisions only for the port site to reduce the high morbidity associated with the laparoscopic procedure as compared to open surgery. METHODS: Between April 2008 and July 2011, we treated 16 consecutive patients (median age, 83 years) with fecal peritonitis resulting from perforations in the left-sided colon due to various causes. The American Society of Anesthesiologists score of each patient was either IV or V. Patients underwent a four-port laparoscopic Hartmann's procedure. Specimens were extracted through the stoma site. Irrigation of the abdominal cavity with more than 10 L of saline was performed in every case, as was insertion of three 10-mm silicon drains via the port site into the left- and right subphrenic spaces or the pouch of Douglas. RESULTS: The median total surgical time was 166 min (range, 123-250 min). There were no intraoperative complications, and there was no need to convert to open surgery. Fourteen patients survived. There was no wound infection or dehiscence of abdominal fascia. Successful laparoscopic reversals of the laparoscopic Hartmann's procedure were performed in all 14 survivors. CONCLUSIONS: This laparoscopic Hartmann's procedure is a promising surgical strategy for treating fecal peritonitis arising from perforation of the left-sided colon.

Successful living donor left liver transplantation by using liver graft with multiple large cysts: a case report.

There are few reports regarding the use of liver grafts with multiple large cysts in living donor liver transplantation. A 40-year-old woman who was diagnosed with Wilson's disease underwent living donor left liver transplantation; the donor was her 67-year-old mother. The liver graft had multiple large cysts, with a maximum diameter of 9 cm. At donor hepatectomy, the largest cyst and one small cyst were fenestrated, because they were located in the left paramedian sector; the other cysts were left intact. After transplantation, the liver graft exhibited good function with no cyst-related complications, such as hemorrhage, infection, or rupture, despite slight enlargement of the cysts. Thus, a liver graft with multiple large cysts is transplantable. However, the necessity of treating large cysts remains debatable.

Studies on the mechanisms responsible for the formation of focal swellings on neuronal processes using a novel in vitro model of axonal injury.

A novel in vitro model of axonal injury using PC12 cells was designed to introduce traumatic alterations on neuronal processes and to identify mechanisms responsible for the formation of focal swellings by observation with phase-contrast and transmission electron microscopes. The injury on the processes was produced by one-dimensional, horizontal oscillation. The fluid shear stress applied by the oscillation did not exceed 380 dyne/cm2. The injured processes showed two forms. One involved an increase in the terminal diameter of the processes and the other entailed beading along the injured portions. Long-term observation of cellular responses to the mechanical insult disclosed that the terminal swelling coincided with the detachment of growth cones from the culture plate. The finding suggests that the detachment of the growth cone destroys the cytoskeletal network, which determines and maintains the cell shape, resulting in spherical deformation of the processes. When the cytoskeletal destruction occurred at non-terminal sites along the processes, spherical deformations developed slowly, and these appeared as beads. The beading also caused the detachment of the growth cones. As the most proximal bead grew, they absorbed the distal segment and their growth cones were pulled proximally with the spreading cytoskeletal destruction. The processes with terminal swellings as well as the bead segments showed regeneration with time evidence of and growth cone formation.

Forced oral opening for cadavers with rigor mortis: two approaches for the myotomy on the temporal muscles.

Forensic dentistry plays an essential role in personal identification procedures. An adequate interincisal space of cadavers with rigor mortis is required to obtain detailed dental findings. We have developed intraoral and two directional approaches, for myotomy of the temporal muscles. The intraoral approach, in which the temporalis was dissected with scissors inserted via an intraoral incision, was adopted for elderly cadavers, females and emaciated or exhausted bodies, and had a merit of no incision on the face. The two directional approach, in which myotomy was performed with thread-wire saw from behind and with scissors via the intraoral incision, was designed for male muscular youths. Both approaches were effective to obtain a desired degree of an interincisal opening without facial damage.

Application of quantitative ethanol detector (QED) test kit to measure ethanol concentration in blood samples.

In this paper, the applicability of the quantitative ethanol detector (QED) test kit for screening of ethanol concentrations in blood samples was investigated. The pretreatment of blood using the sulfosalicylic acid solution and the three-way stopcock followed by membrane filtration gave satisfactory results. The ethanol concentrations in whole blood samples (n=61) determined by QED correlated well with those determined by gas chromatography; the correlation coefficient indicated 0.990. Because a high correlation coefficient (0.928) was also confirmed in trial by investigators, QED test should be highly considered for ethanol screening in forensic praxis.

[Application of Q.E.D. and Alco-Screen test kits to measurements of ethanol in forensic samples].

We have investigated the applicability of the Q.E.D. (Quantitative Ethanol Detector) and Aloco-Screen test kits for screening ethanol concentrations in forensic samples, such as hemolyzed/decomposed blood, urine and vitreous humor. Because both kits were based on enzymatic color reactions, direct application of the kits to hemoglobin-rich samples gave unsatisfactory results. The deproteinization of blood with trichloroacetic acid followed by membrane filtration overcame such problem. This procedure was also effective for pretreatment of urine and vitreous humor samples to suppress excessive color development in the Alco-Screen test. The ethanol concentrations in whole blood (n = 29), urine (n = 7) and vitreous humor (n = 6) samples determined by the Q.E.D. kit correlated well with those determined by gas chromatography; the correlation coefficients were 0.986, 0.975 and 0.993, respectively. Because of its high specificity and sensitivity to ethanol, Q.E.D. seems to be highly reliable for quantitative estimation of ethanol concentrations in forensic samples. Alco-Screen also had high sensitivity, the specificity to ethanol was relatively low; the color reaction was also observed in the presence of acetone, n-propanol, toluene, methanol, ethylene glycol, methamphetamine, diazepam and dichrovos. Therefore, if forensic samples are analyzed by the Alco-Screen, it is essential to confirm the positive results using other analytical methods.

[Pulmonary complications after allogeneic hematopoietic stem cell transplantation].

We investigated the occurrence of pulmonary complications in patients who underwent allogeneic hematopoietic stem cell transplantation at our institution. Pulmonary complications were observed in 12 out of 60 patients. Interstitial pneumonia developed in 12 cases: 7 idiopathic, 2 cytomegalovirus-associated, 1 P. carinii, 1 HSV, and 1 HHV-6-associated. HSV- and HHV-6-associated pneumonias were exhibited 100 days after transplantation. PCR analysis was diagnostically useful for detection of viral DNA in bronchial alveolar lavage fluid. Respiratory disease with airway obstruction was observed in 4 patients with chronic graft-versus-host disease, and all 4 had a history of interstitial pneumonia. Three patients died of respiratory failure. Mycobacicrium avium complex was detected in 2. Exacerbation of respiratory failure may be associated with mycobacterial infection.

Modulation of allogeneic immune responses by filgrastim (recombinant human granulocyte colony-stimulating factor) in bone marrow transplantation.

We examined whether the use of G-CSF would affect the outcome of allogeneic marrow transplantation in humans and mice. Retrospective analysis of 24 patients who had received allogeneic marrow grafts from HLA-identical siblings revealed that the incidence of chronic but not acute graft-versus-host disease (GVHD) was lower in the patients receiving G-CSF than in those not given G-CSF (18% vs. 80%, P = 0.02). There was a difference in serum TNF-alpha levels during the first 3 months after transplant between these two groups. Four out of the ten patients who were not given G-CSF showed elevated serum TNF-alpha levels, whereas there was only one patients with an increased TNF-alpha level among eleven patients who were given G-CSF. With the use of murine acute and chronic GVHD models, we also observed that administration of G-CSF improved the survival of minor GVHD, but not major GVHD, mice. Taken together, these findings suggest that G-CSF down-regulates allogeneic immune responses and is active in modulating alloreactivity in vivo.

Autoantibodies against 70-kDa heat shock proteins (HSP70) in allogeneic bone marrow transplant recipients.

We report that autoantibodies against the 70-kDa heat shock protein family (HSP70) were detected in allogeneic bone marrow transplant recipients. Antibodies to HSP70 family proteins were detected in three out of 14 recipients of an allogeneic marrow graft but in none of the seven patients receiving autologous peripheral blood stem cell transplant (PBSCT). Immunoblotting analysis combined with two-dimensional SDS-PAGE revealed that these patients had antibodies to a constitutive 73-kDa/pI 5.5 heat shock protein (HSP73) and to a stress-inducible 72-kDa/pI 5.6 protein (HSP72). This is the first report, to our knowledge, describing the presence of autoantibody against HSP73 in allogeneic marrow transplant recipients. Our results may provide additional insight into the etiology and the pathophysiology of allogeneic transplant-related disorders.

Granulocyte colony-stimulating factor downregulates allogeneic immune responses by posttranscriptional inhibition of tumor necrosis factor-alpha production.

We report downregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic immune responses in vitro. G-CSF did not affect the proliferative response of peripheral blood mononuclear cells (PBMC) against allogeneic Daudi cells but did inhibit tumor necrosis factor (TNF)-alpha secretion. In contrast with G-CSF, granulocyte-macrophage (GM)-CSF and interleukin (IL)-3 enhanced alloactivation-induced TNF-alpha production. G-CSF-mediated suppression of TNF-alpha production was not affected by fixation of stimulators. G-CSF did not inhibit TNF-alpha mRNA expression or accelerate mRNA degradation, whereas pentoxifylline inhibited the expression of TNF-alpha mRNA. These results indicate that G-CSF acts directly on responder cells and modulates TNF-alpha production at posttranscriptional levels. Suppression of TNF-alpha secretion was accompanied by an increase of intracellular cyclic adenosine monophosphate (cAMP) concentration in alloactivated PBMC. The cell-permeable cAMP analogue, dibutyryl cAMP, suppressed TNF-alpha secretion without affecting TNF-alpha mRNA expression. G-CSF showed an inhibitory effect on the development of cytotoxic effector cells against allogeneic Daudi cells. Anti-TNF-alpha monoclonal antibody (MoAb) also inhibited the induction of cytolytic activity, and the inhibitory effects of G-CSF and anti-TNF-alpha MoAb on killer activity generation were overcome by adding exogenous TNF-alpha. Hence, impaired generation of cytolytic effector cells by G-CSF is believed to be the result of reduced TNF-alpha production. Collectively, the results described above suggest that G-CSF downregulates allogeneic immune responses by posttranscriptionally inhibiting TNF-alpha production.

Successful hematopoietic reconstitution with granulocyte colony-stimulating factor in a patient with hypoplastic acute myelogenous leukemia.

We describe a 68-year-old Japanese male with hypoplastic acute myelogenous leukemia (AML) who achieved complete hematological reconstitution following granulocyte colony-stimulating factor (G-CSF) administration. The patient had pancytopenia and the bone marrow was hypocellular with 19 to 36% peroxidase-positive blasts without morphological abnormalities suggestive of myelodysplasia. After receiving G-CSF as a supportive therapy for pneumonia, the blood count became normal and the bone marrow was normocellular with less than 5% of blasts. Without subsequent chemotherapy, he relapsed as a form of overt leukemia and died of pneumonia. Chemotherapy may be necessary to maintain remission in hypoplastic AML after hematopoietic reconstitution by G-CSF.

Non-genetic group specific component (GC) observed in a patient with acute non-lymphocytic leukemia.

This is the case of a male non-lymphocytic leukemia patient who continuously exhibited non-genetic group specific component (GC). The patient was hospitalized 10 months for treatment. The patient's GC type was GC 2-1A2 upon his admission to the hospital, which was consistent with his parents' type. The additional GC protein isoform appeared during treatment and was detected by polyacrylamide gel isoelectric focusing (PAGIEF). This acquired GC component corresponded to GC 1F in PAGIEF and was thought to have originated from GC 1A2 protein. This phenomenon most probably occurred due to a metabolic abnormality or modification of the GC molecule caused by the disease itself, or by chemotherapy.

Risk factors for hepatosplenic abscesses in patients with acute leukemia receiving empiric azole treatment.

The authors retrospectively evaluated 63 febrile neutropenic episodes in 33 consecutive patients with leukemia who received empiric azole treatment for refractory or relapsing fever that occurred despite broad-spectrum antibiotics. In 8 patients (24%), hepatosplenic abscesses (HSA) developed. To identify the risk factors for the development of HSA, the authors compared various characteristics of febrile episodes in those with and without HSA. The risk factors included relapsed status of leukemia (P = 0.04) and Candida colonization of surveillance cultures from the throat (P = 0.03) and stool (P = 0.03). However, the duration of neutropenia, gastrointestinal symptoms, types of chemotherapy, and leukemia subtypes were not correlated with the development of HSA. Based on these results, the authors identified the high risk group for the development for HSA as patients with relapsed leukemia with fungal colonization of gastrointestinal tract during neutropenia despite empiric antifungal treatment with azoles.

Successful vidarabine therapy for adenovirus type 11-associated acute hemorrhagic cystitis after allogeneic bone marrow transplantation.

A 36-year-old man underwent an allogeneic BMT for acute lymphoblastic leukemia. Eighteen days later the patient developed sudden onset of painful, gross hematuria due to adenovirus type 11 infection that did not respond to conservative therapy. There was an increase in serum creatinine levels and delayed recovery of the platelet count, associated with hemophagocytosis. After obtaining informed consent, vidarabine, which has been shown to be active against adenovirus in vitro, was started. The patient's symptoms improved within a few days of vidarabine therapy and urine cultures for adenovirus became negative. No serious adverse effects were observed. Vidarabine may be one therapeutic option in life-threatening adenovirus infection.

[Acute monoblastic leukemia (M5a) with dysmegakaryocytopoiesis associated with t(16;21) (p11;q22)].

We report a 24-year-old woman who had acute monoblastic leukemia associated with t(16;21) (p11;q22). She was referred to our hospital in April 1992 because of high fever and hemorrhagic diathesis. Physical examination on admission showed no hepatosplenomegaly. The hemoglobin was 5.1g/dl, platelet count 1.7 x 10(4)/microliters, the white blood cell count 18,700/microliters. Bone marrow aspirate showed that 86% of nucleated cells were monoblasts which were positive for peroxidase and alpha-naphtyl butyrate esterase. She was diagnosed as having M5a. Dysmegakaryopoiesis, such as micromegakaryocytes and megakaryocytes with multiple small separated nuclei, was seen in the bone marrow. Chromosomal analysis revealed t(16;21). Complete remission was achieved after two courses of BHAC-DMP therapy, but dysmegakaryopoietic features remained. She relapsed in September 1992. Review of the literature and this patient indicate that acute nonlymphocytic leukemia with t(16;21) is associated with multilineage leukemic differentiation.