Feasibility of transperineal minimal invasive surgery when performing sacrectomy for advanced primary and recurrent pelvic malignancies.

BACKGROUND: This study aimed to clarify the efficacy and safety of minimally invasive transabdominal surgery (MIS) with transperineal minimal invasive surgery (tpMIS) for sacrectomy in advanced primary and recurrent pelvic malignancies. METHODS: Using a prospectively collected database, we retrospectively analyzed the clinical, surgical, and pathological outcomes of MIS with tpMIS for sacrectomies. Surgery was performed between February 2019 and May 2023. The median follow-up period was 27 months (5-46 months). RESULTS: Fifteen consecutive patients were included in this analysis. The diagnoses were as follows: recurrent rectal cancer, n = 11 (73%); primary rectal cancer, n = 3 (20%); and recurrent ovarian cancer, n = 1 (7%). Seven patients (47%) underwent pelvic exenteration with sacrectomy, six patients (40%) underwent abdominoperineal resection (APR) with sacrectomy, and two patients (13%) underwent tumor resection with sacrectomy. The median intraoperative blood loss was 235 ml (range 45-1320 ml). The postoperative complications (Clavien-Dindo grade ≥ 3a) were graded as follows: 3a, n = 6 (40%); 3b, n = 1 (7%); and ≥ 4, n = 0 (0%). Pathological examinations demonstrated that R0 was achieved in 13 patients (87%). During the follow-up period, two patients (13%) developed local re-recurrence due to recurrent cancer. The remaining 13 patients (87%) had no local disease. Fourteen patients (93%) survived. CONCLUSIONS: Although the patient cohort in this study is heterogeneous, MIS with tpMIS was associated with a very small amount of blood loss, a low incidence of severe postoperative complications, and an acceptable R0 resection rate. Further studies are needed to clarify the long-term oncological feasibility.

Feasibility of transanal minimally invasive surgery for total pelvic exenteration for advanced primary and recurrent pelvic malignancies.

BACKGROUND: The purpose of this study was to clarify the efficacy and safety of transanal minimally invasive surgery (TAMIS) for total pelvic exenteration (TPE) in advanced primary and recurrent pelvic malignancies. METHODS: Using a prospectively collected database, we retrospectively analyzed the clinical, surgical, and pathological outcomes of TAMIS for TPE. Surgery was performed between September 2019 and April 2023. The median follow-up period was 22 months (2-45 months). RESULTS: Fifteen consecutive patients were included in this analysis M:F = 14:1 and median (range) age was 63 (36-74). Their diagnoses were as follows: primary rectal cancer (n = 5; 33%), recurrent rectal cancer (n = 4; 27%), primary anorectal cancer (n = 5; 33%), and gastrointestinal stromal tumor (n = 1; 7%). Bladder-sparing TPE was selected for two patients (13%). In nine of 15 patients (60%) the anal sphincter could be successfully preserved, five patients (33%) required combined resection of the internal iliac vessels, and two (13%) required rectus muscle flap reconstruction. The median operative time was 723 min (561-1082), and the median intraoperative blood loss was 195 ml (30-1520). The Clavien-Dindo classifications of the postoperative complications were as follows: grade 0-2 (n = 11; 73%); 3a (n = 3; 20%); 3b (n = 1; 7%); and ≥ 4 (n = 0; 0%). No cases of conversion to laparotomy or mortality were observed. The pathological results demonstrated that R0 was achieved in 14 patients (93%). CONCLUSIONS: The short-term outcomes of this initial experience proved that this novel approach is feasible for TPE, with low blood loss, acceptable postoperative complications, and a satisfactory R0 resection rate.

Alpha-1 antitrypsin limits neutrophil extracellular trap disruption of airway epithelial barrier function.

Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT.

Ostomy creation with fewer sutures using tissue adhesives (cyanoacrylates) in inflammatory bowel disease: a pilot study.

Introduction Fistula formation around the ostomy site is a stoma-related complication often requiring surgical intervention. This complication may be caused by sutures or may develop as a complication of inflammatory bowel disease. Before conducting a clinical trial, we set out to investigate the safety of ostomy creation with fewer sutures using tissue adhesives in this pilot study. Methods Patients with inflammatory bowel disease who required surgery with ostomy creation at the Hyogo College of Medicine between January 2014 and December 2015 were enrolled. Safety was assessed by evaluating the incidence of stoma-related complications. Ostomy was restricted to loop ileostomy and was created with two sutures and tissue adhesives. Results A total of 14 patients were enrolled. Mean body mass index was 18.9 ± 2.0 kg/m2. There were no cases of ostomy retraction and no severe adverse events were observed. Conclusions This pilot study demonstrates that ostomy creation using tissue adhesives is safe. Although retraction and adverse events were not observed, even in patients with inflammatory bowel disease who generally exhibit delayed wound healing, the body mass index was extremely low in this series. This study does not strongly recommend ostomy creation with tissue adhesives; further studies are needed to clarify the efficacy and safety of the procedure.

Prognostic significance of p53 mutations and 3p deletions in primary resected non-small cell lung cancer.

We evaluated the prognostic significance of p53 mutations and an allelic loss of chromosome 3p in 71 patients with non-small cell lung cancer who underwent potentially curative resection. p53 mutations were detected in 35 cases (49%), while 3p deletions were observed in 34 of 70 informative cases (49%). The presence of the p53 mutation was associated with a shortened survival in all patients (P = 0.014 by log rank test), including those in early stages of the disease (stage I or II, n = 48) (P = 0.016 by log rank test). Multivariate analysis by the Cox proportional hazards model also revealed that p53 mutation was an independent yet unfavorable prognostic factor (P = 0.013). Patients with 3p deletion tended to have a poorer prognosis, but not to a statistically significant extent.

Frequent homozygous deletions in lung cancer cell lines detected by a DNA marker located at 3p21.3-p22.

Frequent allelic losses of chromosome 3p in lung cancer have been reported in a number of studies, and we previously demonstrated that 3p21.3 is one of the common regions of deletion in lung cancers and renal cell carcinomas. To further define a region containing the putative tumor suppressor gene, we performed Southern-blot analysis of 26 small cell lung cancer (SCLC) cell lines and ten non-small cell lung cancer (NSCLC) cell lines with 40 cosmid markers located at 3p21.3-p22. One marker detected homozygous deletions of four SCLC cell lines and one NSCLC cell line. None of the other markers revealed homozygous deletions or chromosomal rearrangements in these cell lines. The region of homozygous deletion described here is estimated to consist of less than 1 megabase of DNA, and it is very likely to contain at least one of the tumor suppressor genes associated with carcinogenesis of lung cancer and, possibly, renal cell carcinoma.

Predominantly tumor-limited expression of a mutant allele in a Japanese family carrying a germline p53 mutation.

Germline mutations of p53 have been implicated as a cause of cancer susceptibility in the Li-Fraumeni syndrome. Since inactivation of p53 has been suggested to play an important causative role in lung cancer, the present study of the prevalence of germline mutations in 148 patients with this neoplasm was performed. None of 138 randomly chosen patients were found to carry such mutations, while a single patient had a nonsense mutation at codon 213 among 10 patients selected for early onset and/or occurrence of multiple primary cancers. In contrast to the previous report of biallelic expression of p53 in a case with a germline missense mutation, preferential expression of the wild-type allele was observed in the heterozygous state in both normal lung and peripheral blood lymphocytes of our case, whereas expression of mutant mRNA was readily detectable in her lung cancer in the absence of the remaining wild-type allele. Interestingly, the family history of the proband showed a mild aggregation of adulthood cancers and a high prevalence of stomach cancer, a rare component in American families affected by the syndrome. These observations suggest the presence of heterogeneity with regard to molecular and clinical features of germline p53 mutations.

Aberrant upregulation of a novel integrin alpha subunit gene at 3p21.3 in small cell lung cancer.

Our recent identification of homozygous deletions at 3p21.3 in lung cancer has provided further support for the presence of a tumor suppressor gene in this chromosomal region. As a part of our efforts for positional cloning of a tumor suppressor gene at 3p21.3, we have characterized a transcriptional unit within this region using genomic fragments with interspecies conservation. The identified gene was found to encode a novel integrin alpha subunit, termed alpha RLC, which is closely related to alpha 4 in structure but clearly different from alpha 4 in its expression pattern in the physiological and pathological setting of the lung. This finding and the exact localization of the gene suggest that it is a good candidate for a tumor suppressor gene in lung cancer, but our extensive search covering one third of the gene did not reveal any somatic mutations within the coding region. Interestingly, however, alpha RLC was abundantly expressed in fetal lung and lung cancers, particularly small cell lung cancers (SCLC). Its aberrant upregulation in the SCLC samples, both cell lines and primary tumors, which might have been caused by a yet unidentified mutations or by deletions of other gene, and its homology to alpha 4, which is thought to play a role in metastasis, suggest that altered alpha RLC expression may contribute to the acquisition of malignant phenotypes of this type of lung cancer.

Genetic alteration of the beta-catenin gene (CTNNB1) in human lung cancer and malignant mesothelioma and identification of a new 3p21.3 homozygous deletion.

The beta-catenin gene (CTNNB1) has been shown to be genetically mutated in various human malignancies. To determine whether the beta-catenin gene is responsible for oncogenesis in thoracic malignancies, we searched for the mutation in 166 lung cancers (90 primary tumors and 76 cell lines), one blastoma and 10 malignant mesotheliomas (two primary tumors and eight cell lines). Among the lung cancers, including 43 small cell lung cancers (SCLCs) and 123 non-small cell lung cancers (NSCLCs), we identified four alterations in exon 3, which is the target region of mutation for stabilizing beta-catenin. One primary adenocarcinoma had a somatic mutation from C to G, leading to an amino acid substitution from Ser to Cys at codon 37. Among the cell lines, SCLC NCI-H1092 had a mutation from A to G, leading to an Asp to Gly substitution at codon 6, NSCLC HCC15 had a mutation from C to T, leading to a Ser to Phe substitution at codon 45, and NSCLC NCI-H358 had a mutation from A to G, leading to a Thr to Ala substitution at codon 75. One blastoma also had a somatic mutation from C to G, leading to a Ser to Cys substitution at codon 37. Among the 10 malignant mesotheliomas, we identified a homozygous deletion in the NCI-H28 cell line. Cloning of the rearranged fragment from NCI-H28 indicated that all the exons except exon 1 of the beta-catenin gene are deleted and that the deletion junction is 13 kb downstream from exon 1. Furthermore, Northern blot analysis of 26 lung cancer and eight mesothelioma cell line RNAs detected ubiquitous expression of the beta-catenin messages except NCI-H28, although Western blot analysis showed that relatively less amounts of protein products were expressed in some of lung cancer cell lines. Our findings suggest that the beta-catenin gene is infrequently mutated in lung cancer and that the NCI-H28 homozygous deletion of the beta-catenin gene might indicate the possibility of a new tumor suppressor gene residing in this region at 3p21.3, where various types of human cancers show frequent allelic loss.

Purification of guanylyl cyclase from rod outer segments.

The particulate form of guanylyl cyclase from bovine rod outer segments has been solubilized and purified to near homogeneity by a combination of liquid chromatography and native gel electrophoresis. The procedure enriches enzyme activity 6700-fold from rod outer segment extracts to a final specific activity of 17.5 mumol/min per mg (when assayed with Mn-GTP as substrate). Purified preparations of guanylyl cyclase contain a single glycoprotein with an apparent molecular mass of 60,000 Da and a native isoelectric point of 7.6. Although crude or partially purified enzyme activity is modulated by sub-micromolar concentrations of Ca2+, the fully purified enzyme is insensitive to this cation. However, the purified enzyme remains sensitive to nitrovasodilators, being stimulated over 10-fold by sodium nitroprusside. These data suggest that retinal rods contain a unique isoform of guanylyl cyclase.

Blocking effect of verapamil on conduction over a catecholamine-sensitive bypass tract in exercise-induced Wolff-Parkinson-White syndrome.

By intravenous administration of isoproterenol, 0.5 micrograms/min, a catecholamine-sensitive bypass tract was confirmed in two patients with exercise-induced Wolff-Parkinson-White syndrome. In a 24 year old woman, an intravenous bolus injection of 5 mg of verapamil suddenly blocked conduction over a catecholamine-sensitive bypass tract. In a 62 year old man, the exercise-induced Wolff-Parkinson-White syndrome disappeared after 3 days of oral administration of verapamil (120 mg/day). These observations suggest that a slow inward calcium current plays an important role in conduction over a catecholamine-sensitive bypass tract in exercise-induced Wolff-Parkinson-White syndrome.

LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade.

Malignant mesothelioma (MM) is one of the most aggressive neoplasms usually associated with asbestos exposure and is highly refractory to current therapeutic modalities. MMs show frequent activation of a transcriptional coactivator Yes-associated protein (YAP), which is attributed to the neurofibromatosis type 2 (NF2)-Hippo pathway dysfunction, leading to deregulated cell proliferation and acquisition of a malignant phenotype. However, the whole mechanism of disordered YAP activation in MMs has not yet been well clarified. In the present study, we investigated various components of the NF2-Hippo pathway, and eventually found that MM cells frequently showed downregulation of LIM-domain protein AJUBA, a binding partner of large tumor suppressor type 2 (LATS2), which is one of the last-step kinases of the NF2-Hippo pathway. Although loss of AJUBA expression was independent of the alteration status of other Hippo pathway components, MM cell lines with AJUBA inactivation showed a more dephosphorylated (activated) level of YAP. Immunohistochemical analysis showed frequent downregulation of AJUBA in primary MMs, which was associated with YAP constitutive activation. We found that AJUBA transduction into MM cells significantly suppressed promoter activities of YAP-target genes, and the suppression of YAP activity by AJUBA was remarkably canceled by knockdown of LATS2. In connection with these results, transduction of AJUBA-expressing lentivirus significantly inhibited the proliferation and anchorage-independent growth of the MM cells that harbored ordinary LATS family expression. Taken together, our findings indicate that AJUBA negatively regulates YAP activity through the LATS family, and inactivation of AJUBA is a novel key mechanism in MM cell proliferation.

Analysis of epidermal-type transglutaminase (TGase 3) expression in mouse tissues and cell lines.

In the formation of the cornified cell envelope in the epidermis, epidermal-type transglutaminase (TGase 3) cross-links a variety of structural proteins. However, its expression in other tissue has not been investigated. Furthermore, no cell line expressing TGase 3 has been found. The tissue distribution of TGase 3 in mice was investigated using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting analyses. TGase 3 mRNA was expressed in the brain, stomach, spleen, small intestine, testis, skeletal muscle and skin. The stomach and testis expressed TGase 3 protein in size similar to that observed in the epidermis. Screening various cell lines, a gastric human cancer cell line, MKN-1 and mouse neuroblast cell line, neuro2a, were found to express TGase 3.

Differential distribution of classical inwardly rectifying potassium channel mRNAs in the brain: comparison of IRK2 with IRK1 and IRK3.

Distribution of IRK2 inwardly rectifying potassium channel mRNA in the mouse brain was studied using in situ hybridization histochemistry and compared with those of other classical inwardly rectifying potassium channel (IRK1 and IRK3) mRNAs. All these IRK channel mRNAs were detected in neurons, but not in glial cells. Their distribution patterns in the brain were, however, quite divergent: IRK2 mRNA was detected extremely high in granule cells of cerebellum, relatively high in motor trigeminal nucleus and moderate in olfactory bulb, piriform cortex, cerebral cortex, CA1 through CA3 regions of hippocampus, dentate gyrus and pontine nucleus. On the other hand, IRK1 mRNA was expressed throughout whole brain but in particular subsets of neurons, and IRK3 mRNA was in forebrain. Expression of these three IRK mRNAs overlapped in hippocampus, olfactory bulb, and cerebral cortex. This differential distribution of IRK mRNAs suggests that each of these channels has its specific function in regulation of the excitability of brain neurons.

Somatostatin induces hyperpolarization in pancreatic islet alpha cells by activating a G protein-gated K+ channel.

Somatostatin inhibits glucagon-secretion from pancreatic alpha cells but its underlying mechanism is unknown. In mouse alpha cells, we found that somatostatin induced prominent hyperpolarization by activating a K+ channel, which was unaffected by tolbutamide but prevented by pre-treating the cells with pertussis toxin. The K+ channel was activated by intracellular GTP (with somatostatin), GTPgammaS or Gbetagamma subunits. It was thus identified as a G protein-gated K+ (K(G)) channel. RT-PCR and immunohistochemical analyses suggested the K(G) channel to be composed of Kir3.2c and Kir3.4. This study identified a novel ionic mechanism involved in somatostatin-inhibition of glucagon-secretion from pancreatic alpha cells.

High-resolution immunogold cytochemistry indicates that AQP4 is concentrated along the basal membrane of parietal cell in rat stomach.

Gastric parietal cells secrete hydrochloric acid in stomach. Because the secreted HCl solution is isotonic with the plasma fluid, it should accompany the water transport across the membranes of parietal cells. Aquaporins (AQPs) are water channel proteins that play the central role in the cellular handling of water in various mammalian tissues. Using immunocytochemistry, we found that AQP4 was expressed only in parietal cells of rat gastric mucosa. Immunogold electron microscopy study further demonstrated that AQP4 was mostly localized at the basal membrane of parietal cells. In the basal membrane, AQP4 was prominently enriched on the portion contacting with the basement membrane surrounding gastric glands. These results suggest that the contact between basement membrane and basal membrane may generate the signal involved in the targeting of AQP4 in gastric parietal cells.

Cloning and functional expression of a novel isoform of ROMK inwardly rectifying ATP-dependent K+ channel, ROMK6 (Kir1.1f).

We have identified from rat kidney a novel isoform of ROMK/Kir1.1, designated ROMK6/Kir1.1f. ROMK6 was nearly identical to ROMK1, but possessed an 122-bp insertion in the 5' region. Its deduced amino acid sequence was shorter by 19 amino acids than that of ROMK1 in the amino-terminus. Unlike other previously reported ROMK isoforms, ROMK6 mRNA was ubiquitously expressed in various tissues, including kidney, brain, heart, liver, pancreas and skeletal muscle. Xenopus oocytes injected with ROMK6 cRNA expressed a Ba2+-sensitive weakly inwardly rectifying K+ current. These results indicate that ROMK6 is a novel functional K+ channel and might be involved in K+ secretion in various tissue.

Immunolocalization of an inwardly rectifying K+ channel, K(AB)-2 (Kir4.1), in the basolateral membrane of renal distal tubular epithelia.

Immunolocalization of K(AB)-2 (Kir4.1), an inwardly rectifying K+ channel with a putative ATP-binding domain, was examined in rat kidney where expression of K(AB)-2 mRNA was previously shown. Anti-K(AB)-2 antibody was raised in rabbit and then affinity-purified. An immunohistochemical study revealed that K(AB)-2 immunoreactivity was detected specifically in the basolateral membrane of distal tubular epithelia. Therefore, K(AB)-2 is the first K+ channel shown to be localized in the basolateral membrane of renal epithelia. The finding suggests that K(AB)-2 may contribute to supplying K+ to the Na(+)-K+ pump, which is abundant in the basolateral membrane of distal tubular epithelia, as well as to maintenance of the deep negative membrane potential of these cells.

Synergistic effects of adenovirus expressing wild-type p53 on chemosensitivity of non-small cell lung cancer cells.

The infection of recombinant adenovirus expressing wild-type p53 (Ad-p53) to lung cancer cells that harbor mutant p53 genes improves their response to cis-diamminedichloroplatinum(II). In this study, we tested whether this improvement in response is also seen in wild-type p53 (wt-p53)-containing cancer cells and whether this phenomenon is universal with other commonly used chemotherapeutic agents, including etoposide, 7-ethyl-10-hydrocycamptothecin, paclitaxel, and docetaxel. Using a panel of 7 non-small cell lung cancer cell lines with wild-type (2) or abnormal (2 null, 3 point-mutated) p53, we examined in vitro cytotoxicity using a tetrazolium-based colorimetric assay (3-(4,5-diethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide assay) and analyzed the combined effects of Ad-p53 and chemotherapeutic agents using the isobologram method. Ad-p53 and DNA-damaging agents (cis-diamminedichloroplatinum(II), etoposide, and 7-ethyl-10-hydrocycamptothecin) showed synergistic effects in six of seven cell lines but additive effects against a p53-mutated cell line. In contrast, Ad-p53 showed additive effects with the antitubulin agents (paclitaxel and docetaxel) in all four of the cell lines tested. Furthermore, we examined this synergistic interaction between Ad-p53 and DNA-damaging agents by flow cytometric analysis and DNA fragmentation analysis. Both analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by DNA-damaging agents in six of seven cell lines. Our results suggest that Ad-p53 may synergistically enhance the chemosensitivity of the majority of non-small cell lung cancers to DNA-damaging agents due to augmentation of apoptosis.

Induction of antitumor immunity by transduction of CD40 ligand gene and interferon-gamma gene into lung cancer.

CD40-CD40 ligand (CD40L) interaction is an important costimulatory signaling pathway in the crosstalk between T cells and antigen-presenting cells. This receptor-ligand system is known to be essential in eliciting strong cellular immunity. Here we demonstrate that murine lung cancer cells (3LLSA) transduced with the CD40L gene (3LLSA-CD40L) were rejected in syngeneic C57BL/6 mice, but grew in CD40-deficient mice to the same extent as control tumor cells. Immunohistochemical study showed that inflammatory cells, including CD4+, CD8+ T cells and NK cells, infiltrated into the inoculated 3LLSA-CD40L tumor tissue. Inoculation of 3LLSA-CD40L cells into mice resulted in the induction of 3LLSA-specific cytotoxic T-cell immunity, and the growth of parental 3LLSA tumors was inhibited when 3LLSA cells were inoculated into C57BL/6 mice mixed with 3LLSA-CD40L cells or when they were rechallenged 4 weeks after 3LLSA-CD40L cells were rejected. Furthermore, co-inoculation of interferon (IFN)-gamma-transduced cells (3LLSA-IFNgamma) with 3LLSA-CD40L cells enhanced the antitumor immunity efficiently in vivo. These results indicate that the in vivo priming with CD40L- and IFN-gamma gene-transduced lung cancer cells is a promising strategy for inducing antitumor immunity in the treatment of lung cancer.