Spatiotemporal metabolic dynamics of the photosensitizer talaporfin sodium in carcinoma and sarcoma.

Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co-localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin- and caveolae-dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K-Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin- and caveolae-dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K-Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma.

Upstream Position of Proline Defines Peptide-HLA Class I Repertoire Formation and CD8+ T Cell Responses.

Cytotoxic CD8+ T lymphocytes (CTLs) recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as cancer. Difficulty in predicting HLA class I ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I ligands on a large scale and analyzed the source-protein sequences flanking the ligands. This comprehensive analysis revealed that the frequency of proline at amino acid positions 1-3 upstream of the ligands was selectively decreased. The depleted proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of proline at upstream positions 1-3 attenuated CTL responses against a model epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the proline signature. Our results highlight, to our knowledge, a unique role and position of proline for inhibiting downstream epitope presentation, which provides a rule for defining natural peptide-HLA class I repertoire formation and CTL responses.

Clonal analysis revealed functional heterogeneity in cancer stem-like cell phenotypes in uterine endometrioid adenocarcinoma.

Uterine endometrial carcinoma is one of the common cancers in females. Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are tumorigenic and are resistant to treatments, thus they are focused as treatment targets. However, the heterogeneity of CSCs/CICs is still elusive, and we therefore analyzed CSCs/CICs at the clonal level. We previously established sphere-cultured CSCs/CICs from primary human uterine endometrial carcinoma, and we isolated several clones from CSCs/CICs in this study. Interestingly, we established two types of clones based on the growth pattern. The clones were termed sphere clones (S clones) and leukemia-like clones (LL clones). Functional analysis revealed that S clones are resistant to chemotherapy, whereas LL clones are sensitive to chemotherapy. On the other hand, S clones are less tumorigenic, while LL clones are highly tumorigenic. Transcriptome analysis using serial analysis of gene expression sequencing (SAGE-Seq) revealed distinctive gene expression profiles in S clone cells and LL clone cells. The results indicate that CSCs/CICs are composed of functionally heterogenic subpopulations including highly tumorigenic clones and treatment-resistant clones and that the characteristics of CSCs/CICs might be determined by the characteristics of different clones that compose CSCs/CICs.

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

BACKGROUND/AIM: Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. MATERIALS AND METHODS: Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. RESULTS: Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. CONCLUSION: High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

Laparoscopic approach for surgical treatment of pleuroperitoneal communication interfering with peritoneal dialysis: a case report.

BACKGROUND: Pleuroperitoneal communication is a rare disorder that interferes with peritoneal dialysis. Although favorable results of thoracoscopic fistula closure have been reported, there are some cases in which the fistulas cannot be identified by thoracoscopy and the patients are forced to switch to hemodialysis. CASE PRESENTATION: We present two cases of pleuroperitoneal communication in which diaphragmatic fistulas could not be identified thoracoscopically, but could be identified laparoscopically. Patient 1 had difficulty continuing peritoneal dialysis 9 months after its introduction due to right pleural effusion. Although we could not detect the fistula thoracoscopically, we could laparoscopically identify the fistula in the center of the tendon of the right diaphragm and closed the site from the thoracic side. Patient 2 developed dyspnea due to right pleural effusion 6 months after the introduction of peritoneal dialysis. We could not find the fistulas with a thoracoscopic approach, but could identify multiple diaphragmatic fistulas with a laparoscopic approach and close the sites from the thoracic side. CONCLUSION: In the surgical treatment of pleuroperitoneal communication, diaphragmatic fistulas can be identified laparoscopically even when thoracoscopic observation fails to find any fistulas.

ABCG2 expression is related to low 5-ALA photodynamic diagnosis (PDD) efficacy and cancer stem cell phenotype, and suppression of ABCG2 improves the efficacy of PDD.

Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.

Novel cell-surface peptides specific to human oral squamous cell carcinoma using an E. coli peptide display library.

We attempted to find a specific antigen of oral squamous cell carcinoma (SCC) cells that could be safely applied to gene therapy in the conservative clinical treatment of oral cancer. We performed subtraction using normal human keratinocyte cells, followed by selection using four oral SCC cell lines. We isolated three clones from poorly differentiated SCC cells and four from well-differentiated SCC cells. These seven clones adsorbed to the oral SCC cells at rates 10-100 times those of normal human keratinocyte cells. The three clones from the poorly differentiated SCC cells showed the same peptide sequence (LAPRTHP). Of the four clones from the well-differentiated SCC cells, three showed the same peptide sequence (FGTLPGT) and the fourth showed a different one (VTPNSTP). Each peptide sequence may recognize the material that exists specifically on the oral SCC cell cortex. We can expect applications not only for tumor-targeting treatment using a gene therapy virus vector but also for diagnosis using, as a tumor marker, the peculiar SCC surface material that these peptides recognize.

Usefulness of contrast enhanced-MRI in the diagnosis of unicystic ameloblastoma.

The radiographic features of unicystic ameloblastoma (UA) are typically unilocular and round area of radiolucency. Therefore, this type of lesion is often misdiagnosed as an odontogenic keratocyst or a dentigerous cyst. UA should be differentiated from odontogenic cysts because the former have a higher rate of recurrence than the latter. In the present study, we performed contrast enhanced-MRI (CE-MRI) to diagnose 13 cases of unilocular, round radiolucent lesions visualized on panoramic radiograph and/or CT. In the cases of UA, low signal intensity was observed on T1-weighted images (WIs), and a markedly high signal intensity was observed on T2-WIs; moreover, relatively thick rim-enhancement with/without small intraluminal nodules was observed upon CE-T1WIs. CE-MRI was considered useful in the diagnosis of UA, as characteristic features of this type of lesion i.e., thick enhancement of the tumor wall and small intraluminal nodules were detected only by CE-MRI in the present study.

Effects of histone deacetylase inhibitor FR901228 on the expression level of telomerase reverse transcriptase in oral cancer.

We speculated whether or not the expression level of telomerase reverse transcriptase (hTERT) would be modulated by agents targeting epigenetics in oral cancer cell lines. Although hTERT is known to be targeted by epigenetic changes, it remains unclear how chemoagents targeting epigenetics work on hTERT transcription. In the present study, the epigenetic effects of the histone deacetylase (HDAC) inhibitor FR901228 on hTERT transcription in oral cancer cell lines were analyzed by RT-PCR. The mRNA expression of hTERT was upregulated after exposure to FR901228 in hTERT-negative Hep2 cells, and even SAS and KB cells expressed high levels of hTERT. Moreover, cotreatment of protein synthesis inhibitor cycloheximide (CHX) resulted in the induction of hTERT transcription by FR901228. This suggests that the induction of hTERT by FR901228 requires de novo protein synthesis to some extent and is more likely a direct than an indirect effect on epigenetic changes such as histone acetylation/deacetylation. We further examined the effect of FR901228 on c-myc protein, which is one of the main hTERT transcription activators. FR901228 repressed c-myc protein only in the absence of CHX, and depended on the enhancement of de novo protein synthesis. Our results indicate that c-myc protein is repressed indirectly by FR901228 but may not contribute to FR901228-induced hTERT transcription. The present study showed that the HDAC inhibitor FR901228 induced the hTERT gene by a complex mechanism that involved transcription factors other than c-myc, in addition to inhibition of histone deacetylation.

Role of O6-methylguanine-DNA methyltransferase and effect of O6-benzylguanine on the anti-tumor activity of cis-diaminedichloroplatinum(II) in oral cancer cell lines.

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) modulates the effectiveness of alkylating agents. However, the relationship between MGMT and the sensitivities to other agents has not been explored. In the present study, the association between MGMT expression and the cellular sensitivity to the platinum agent, CDDP was examined in four human oral cancer cell lines. CDDP depleted MGMT protein and mRNA levels in all four cell lines. Two cell lines with low MGMT expression were sensitive to an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine and CDDP, whereas two other cell lines with high MGMT expression were resistant to both agents. Furthermore, the addition of the MGMT inhibitor, O6-benzylguanine (O6-BG), invariably enhanced CDDP sensitivity. CDDP depleted MGMT expression, and CDDP sensitivity was enhanced by O6-BG. These results provide valuable information about the relationship between MGMT expression and CDDP sensitivity in oral cancer chemotherapy.

Assessment of ameloblastomas using MRI and dynamic contrast-enhanced MRI.

We retrospectively evaluated magnetic resonance images (MRI) and dynamic contrast-enhanced MRI (DCE-MRI) of ameloblastomas. MRI and DCE-MRI were performed for 10 ameloblastomas. We obtained the following results from the MRI and DCE-MRI. (a) Ameloblastomas can be divided into solid and cystic portions on the basis of MR signal intensities. (b) Ameloblastomas show a predilection for intermediate signal intensity on T1WI, high signal intensity on T2WI, and well enhancement in the solid portion; they also show a homogeneous intermediate signal intensity on T1WI and homogeneous high signal intensity on T2WI, and no enhancement in the cystic portion. (c) The mural nodule or thick wall can be detected in ameloblastomas lesions. (d) CI curves of ameloblastomas show two patterns: the first pattern increases, reaches a plateau at 100-300 s, then sustains the plateau or decreases gradually to 600-900 s, while the other increases relatively rapidly, reaches a plateau at 90-120 s, then decreases relatively rapidly to 300 s, and decreases gradually thereafter. There was no difference in the CI curve patterns among primary and recurrent cases, a case with glandular odontogenic tumor in ameloblastoma or among histopathological types such as plexiform, follicular, mixed, desmoplastic, and unicystic type.

[Creation and evaluation of the respective rank problem solution worksheet for improvement pharmaceutical management quality].

A respective rank problem solution worksheet was developed focusing on problem solution in training in pharmaceutical management. How the training influenced the instructions given to patients was then evaluated in two pharmacists in the NTT East Kanto Medical Center and three in an Ofuna central hospital. After the five pharmacists underwent the training, the records of 10 medication instructions to patients given by each before and after the training were compared. The records were analyzed based on a point calculation table, and the number of acquisition points was computed. The acquisition points increased significantly after training for all five pharmacists, from a mean of 3.56 before training to a mean of 8.34 after training. Although the acquisition points related to patient education were high, those for intervention during therapeutic monitoring, such as for adverse drug reaction or the selection of appropriate pharmaceuticals were acceptable, increasing to a mean 8.4 items after training compared with 4.4 items before. The respective rank problem solution worksheet is therefore considered useful in improving the quality of training pharmaceutical management.

[The association between dispensing error factors and behavioral characteristics of pharmacists].

We evaluated error prevention education by clarifying the association between dispensing error factors and behavioral characteristics of pharmacists. The subjects were 98 pharmacists (27 men and 71 women) with a mean age of 29.7 years who gave informed consent for participation in our survey. Between November 2001 and January 2002, a questionnaire survey on dispensing errors was performed using the "Tokyo University Egogram, New Version" for the assessment of behavioral characteristics and the "Safety Behavior Questionnaire" for the assessment of error factors. An association was observed between the incidence of dispensing errors and behavioral characteristics. There was also an association between error contents and behavioral characteristics as well as error factors. With more experience, errors associated with becoming accustomed increased, suggesting that error prevention education is necessary not only for newly qualified pharmacists but also for managers.

[Effect of pharmaceutical care in the diagnosis of Helicobacter pylori infection using 13C-urea breath test].

The urea breath test (UBT) is used widely for assessment of Helicobacter pylori (H. pylori) eradication after treatment. A false-negative UBT is common during administration of anti-ulcer drugs and immediately after their discontinuation. It was thought that the pharmaceutical care by the pharmacists was necessary for the diagnostic accuracy of UBT after H. pylori eradication therapy. Therefore, we investigated the effect of pharmaceutical care on diagnosis based on assessment of UBT. The patients who performed UBT were classified into two groups according to the pharmacists' intervention. From 2008 April to 2009 September, the number of the patients taken pharmaceutical care was 57 (intervention group) and that of the patients taken no pharmaceutical care was 62 (control group). When drugs for H. pylori infection and anamnestic therapy were same, the percentage that avoided administration of double drugs was significantly increased by the pharmaceutical care (93.3% in intervention group versus 21.4% in control group, p<0.05). Therefore, the percentage of noncompliance that performed UBT 4 weeks after treatment onward was significantly decreased by the pharmaceutical care (1.6% in intervention group versus 17.5% in control group, p<0.05). Moreover, the percentage of recurrence after treatment was significantly decreased, there were 3.3% in the intervention group and 14.0% in the control group. In conclusion, it was very important that the pharmacists take care in the management of treatment and UBT for H. pylori eradication therapy.

Bone morphogenetic protein (BMP) type II receptor is required for BMP-mediated growth arrest and differentiation in pulmonary artery smooth muscle cells.

Bone morphogenetic protein (BMP) signals regulate the growth and differentiation of diverse lineages. The association of mutations in the BMP type II receptor (BMPRII) with idiopathic pulmonary arterial hypertension suggests an important role of this receptor in vascular remodeling. Pulmonary artery smooth muscle cells lacking BMPRII can transduce BMP signals using ActRIIa (Activin type II receptor). We investigated whether or not BMP signaling via the two receptors leads to differential effects on vascular smooth muscle cells. BMP4, but not BMP7, inhibited platelet-derived growth factor-activated proliferation in wild-type pulmonary artery smooth muscle cells, whereas neither ligand inhibited the growth of BMPRII-deficient cells. Adenoviral gene transfer of BMPRII enabled BMP4, as well as BMP7, to inhibit proliferation in BMPRII-deficient cells. BMP-mediated growth inhibition was also reconstituted by the BMPRII short isoform, lacking the C-terminal domain present in the long form. BMP4, but not BMP7, induced the expression of osteoblast markers in wild-type cells, whereas neither ligand induced these markers in BMPRII-deficient cells. Overexpression of short or long forms of BMPRII in BMPRII-deficient cells enabled BMP4 and BMP7 to induce osteogenic differentiation. Although signaling via BMPRII or ActRIIa transiently activated SMAD1/5/8, only BMPRII signaling led to persistent SMAD1/5/8 activation and sustained increases in Id1 mRNA and protein expression. Pharmacologic blockade of BMP type I receptor function within 24 h after BMP stimulation abrogated differentiation. These data suggest that sustained BMP pathway activation, such as that mediated by BMPRII, is necessary for growth and differentiation control in vascular smooth muscle.

Bone morphogenetic protein (BMP) type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells.

Bone morphogenetic protein (BMP) ligands signal by binding the BMP type II receptor (BMPR2) or the activin type II receptors (ActRIIa and ActRIIb) in conjunction with type I receptors to activate SMADs 1, 5, and 8, as well as members of the mitogen-activated protein kinase family. Loss-of-function mutations in Bmpr2 have been implicated in tumorigenesis and in the etiology of primary pulmonary hypertension. Because several different type II receptors are known to recognize BMP ligands, the specific contribution of BMPR2 to BMP signaling is not defined. Here we report that the ablation of Bmpr2 in pulmonary artery smooth muscle cells, using an ex vivo conditional knock-out (Cre-lox) approach, as well as small interfering RNA specific for Bmpr2, does not abolish BMP signaling. Disruption of Bmpr2 leads to diminished signaling by BMP2 and BMP4 and augmented signaling by BMP6 and BMP7. Using small interfering RNAs to inhibit the expression of other BMP receptors, we found that wild-type cells transduce BMP signals via BMPR2, whereas BMPR2-deficient cells transduce BMP signals via ActRIIa in conjunction with a set of type I receptors distinct from those utilized by BMPR2. These findings suggest that disruption of Bmpr2 leads to the net gain of signaling by some, but not all, BMP ligands via the activation of ActRIIa.

Reactive oxygen species scavengers attenuate endotoxin-induced impairment of hypoxic pulmonary vasoconstriction in mice.

BACKGROUND: Sepsis and endotoxemia attenuate hypoxic pulmonary vasoconstriction (HPV), thereby impairing systemic oxygenation. Reactive oxygen species (ROS) are implicated in the pathogenesis of sepsis-induced lung injury. The authors investigated whether treatment with scavengers of ROS prevents impairment of HPV in mice challenged with endotoxin. METHODS: The pulmonary vasoconstrictor response to left mainstem bronchus occlusion (LMBO) was studied in anesthetized mice 22 h after an intraperitoneal challenge with saline solution or 10 mg/kg Escherichia coli endotoxin. In some mice, challenge with saline solution or endotoxin was followed after 1 h with intraperitoneal or intratracheal administration of the ROS scavengers N-acetylcysteine or EUK-8. Myeloperoxidase activity and nitric oxide synthase-2 gene expression were measured in lung tissues. RESULTS: The LMBO increased left pulmonary vascular resistance by 106 +/- 24% in saline-challenged control mice but by only 23 +/- 12% (P < 0.05) in endotoxin-challenged mice. Intraperitoneal administration of N-acetylcysteine or EUK-8 1 h after endotoxin challenge attenuated the endotoxin-induced impairment of HPV (58 +/- 6% and 68 +/- 10%, respectively; both P< 0.05 endotoxin-challenged mice). Intratracheal administration of ROS scavengers 1 h after endotoxin challenge was equally effective but required lower doses than systemic treatment. Administration of the ROS scavengers 22 h after endotoxin challenge did not restore HPV. CONCLUSIONS: Administration of N-acetylcysteine or EUK-8 1 h after endotoxin challenge in mice prevented the impairment of HPV after LMBO. Early therapy with ROS scavengers, either systemically or by inhalation, may provide a means to preserve HPV in sepsis-associated acute lung injury.

BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia.

Heterozygous mutations of the bone morphogenetic protein type II receptor (BMPR-II) gene have been identified in patients with primary pulmonary hypertension. The mechanisms by which these mutations contribute to the pathogenesis of primary pulmonary hypertension are not fully elucidated. To assess the impact of a heterozygous mutation of the BMPR-II gene on the pulmonary vasculature, we studied mice carrying a mutant BMPR-II allele lacking exons 4 and 5 (BMPR-II(+/-) mice). BMPR-II(+/-) mice had increased mean pulmonary arterial pressure and pulmonary vascular resistance compared with their wild-type littermates. Histological analyses revealed that the wall thickness of muscularized pulmonary arteries (<100 mum in diameter) and the number of alveolar-capillary units were greater in BMPR-II(+/-) than in wild-type mice. Breathing 11% oxygen for 3 wk increased mean pulmonary arterial pressure, pulmonary vascular resistance, and hemoglobin concentration to similar levels in BMPR-II(+/-) and wild-type mice, but the degree of muscularization of small pulmonary arteries and formation of alveolar-capillary units were reduced in BMPR-II(+/-) mice. Our results suggest that, in mice, mutation of one copy of the BMPR-II gene causes pulmonary hypertension but impairs the ability of the pulmonary vasculature to remodel in response to prolonged hypoxic breathing.

A selective inducible NOS dimerization inhibitor prevents systemic, cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice.

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.