Clinical value of R-spondins in triple-negative and metaplastic breast cancers.

BACKGROUND: RSPO ligands, activators of the Wnt/ß-catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC). METHODS: Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT-PCR. The effect of RSPO on the Wnt/ß-catenin pathway activity was determined by luciferase assay, western blotting, and qRT-PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/ß-catenin pathway, was examined on the growth of an RSPO2-positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC. RESULTS: We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO, and amplification or hypomethylation of RSPO genes. Patients with RSPO2-overexpressing tumours have a poorer metastasis-free survival (P=3.6 × 10-4). RSPO2 and RSPO4 stimulate Wnt/ß-catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2-overexpressing PDX. CONCLUSIONS: RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

Effect of positive end-expiratory pressure on pulmonary shunt and dynamic compliance during abdominal surgery.

BACKGROUND: General anaesthesia decreases pulmonary compliance and increases pulmonary shunt due to the development of atelectasis. The presence of capnoperitoneum during laparoscopic surgery may further decrease functional residual capacity, promoting an increased amount of atelectasis compared with laparotomy. The aim of this study was to evaluate the effects of different levels of positive end-expiratory pressure (PEEP) in both types of surgery and to investigate whether higher levels of PEEP should be used during laparoscopic surgery. METHODS: This prospective observational study included 52 patients undergoing either laparotomy or laparoscopic surgery. Three levels of PEEP were applied in random order: (1) zero (ZEEP), (2) 5 cmH2O and (3) 10 cmH2O. Pulmonary shunt and ventilation/perfusion mismatch were assessed by the automatic lung parameter estimator system. RESULTS: Pulmonary shunt was similar in both groups. However, in laparotomy, a PEEP of 5 cmH2O significantly decreased shunt when compared with ZEEP (12 vs 6%; P=0.001), with additional PEEP having no further effect. In laparoscopic surgery, a significant reduction in shunt (13 vs 6%; P=0.001) was obtained only at a PEEP of 10 cmH2O. Although laparoscopic surgery was associated with a lower pulmonary compliance, increasing levels of PEEP were able to ameliorate it in both groups. CONCLUSION: Both surgeries have similar negative effects on pulmonary shunt, while the presence of capnoperitoneum reduced only the pulmonary compliance. It appears that a more aggressive PEEP level is required to reduce shunt and to maximize compliance in case of laparoscopic surgery.

A short-term colorectal cancer sphere culture as a relevant tool for human cancer biology investigation.

BACKGROUND: Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool. METHODS: Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT-PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies. RESULTS: Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell-cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts. CONCLUSION: Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models.

Shear wave elastography of tumour growth in a human breast cancer model with pathological correlation.

OBJECTIVE: To assess stiffness in a human breast cancer implanted in mice using shear wave elastography (SWE) during tumour growth and to correlate the results with pathology. METHODS: Local ethics committee for animal research approval was obtained. A human invasive ductal carcinoma was implanted subcutaneously in 24 athymic nude female mice. Ultrasound was longitudinally performed in 22 tumours, every 1-2 weeks. Maximum diameter and mean stiffness were collected. Seven tumours were measured both in vivo and ex vivo. Tumours of different sizes were removed for pathological analysis on which the percentages of viable cellular tissue, fibrosis and necrosis were measured. RESULTS: A total of 63 SWE measurements were performed. Stiffness increased during tumour growth with an excellent correlation with size (r = 0.94, P < 0.0001). No differences were found between the values of stiffness in vivo and ex vivo (P = 0.81). There was a significant correlation between elasticity and fibrosis (r = 0.83, P < 0.0001), a negative correlation with necrosis (r = -0.76, p = 0.0004) but no significant correlation with cellular tissue (r = 0.40, p = 0.1). CONCLUSION: Fibrosis plays an important role in stiffness as measured by SWE, whereas necrosis is correlated with softness. KEY POINTS: • In a breast cancer model, ultrasound tumour stiffness is correlated with size. • Stiffness changes with tumour growth are correlated with pathological changes. • Stiffness is very well correlated with proportion of tumour fibrosis. • Stiffness is inversely correlated with proportion of tumour necrosis. • Tumour stiffness measurements are similar in vivo and ex vivo.

Modeling of response to endocrine therapy in a panel of human luminal breast cancer xenografts.

Resistance to endocrine therapy is a major complication of luminal breast cancer and studies of the biological features of hormonal resistance are limited by the lack of adequate preclinical models. The aim of this study is to establish and characterize a panel of primary human luminal breast carcinoma xenografts, and to evaluate their response to endocrine therapies. Four hundred and twenty-three tumor fragments obtained directly from patients have been grafted in the interscapular fatpad of Swiss nude mice. After stable engraftment with estradiol supplementation, xenografted tumors have been validated by conventional pathology and immunohistochemistry examination, and additional molecular studies. In vivo tumor growth and response to different endocrine treatments were evaluated. We have engrafted 423 tumors including 314 ER+ tumors, and 8 new luminal breast cancer xenografts have been obtained (2.5%). Tumor take was much lower for luminal tumors than for non-luminal tumors (2.5 vs. 24.7%, P < 0.0001), and was associated with two independent criteria, i.e., ER status (P < 0.0001) and a high grade tumor (P = 0.05). Histological and immunohistochemical analyses performed on patient's tumors and xenografts showed striking similarities in the tumor morphology as well as in the expression level of ER, PR, and HER2. Response to hormone therapy, evaluated in 6 luminal models, showed different sensitivities, thus exhibiting heterogeneity similar to what is observed in the clinic. We have established a panel of primary human luminal breast cancer xenografts, recapitulating the biological and clinical behaviors of patient tumors, and therefore suitable for further preclinical experiments.

Recurrent pneumothorax after cesarean delivery in the critically ill pregnant with severe COVID-19 ARDS: a case report.

OBJECTIVE: Few data are available on the ICU management and on the possible respiratory complications of invasively ventilated pregnant patients affected by COVID-19 pneumonia, especially in the early phase of pregnancy. Tension pneumothorax has been previously described as a rare cause of respiratory failure after delivery, but its occurrence in the postpartum of COVID-19 patient has not been reported yet. We hereby describe the ICU management of a 23rd gestational week pregnant woman who underwent invasive mechanical ventilation, prone positioning, and cesarean delivery during her ICU stay for COVID-19 related pneumonia. Moreover, we focused on the occurrence and management of recurrent tension pneumothorax after the cesarean delivery. CASE REPORT: A 23rd gestational week pregnant woman was admitted to the ICU for a COVID-19 bilateral pneumonia and underwent invasive mechanical ventilation and prone positioning. Cesarean delivery was planned during the ICU stay, while the patient was receiving invasive mechanical ventilation. After delivery, the patient experienced a recurrent pneumothorax that required the positioning of multiple chest drains. CONCLUSIONS: In pregnant critically ill COVID-19 patients, mechanical ventilation management is particularly challenging, especially in the postpartum period. Prone positioning is feasible and can improve oxygenation and respiratory system compliance, while tension pneumothorax must be suspected if the respiratory function suddenly deteriorates after delivery.

Establishment and characterisation of a new breast cancer xenograft obtained from a woman carrying a germline BRCA2 mutation.

BACKGROUND: The BRCA2 gene is responsible for a high number of hereditary breast and ovarian cancers, and studies of the BRCA2 biological functions are limited by the lack of models that resemble the patient's tumour features. The aim of this study was to establish and characterise a new human breast carcinoma xenograft obtained from a woman carrying a germline BRCA2 mutation. METHODS: A transplantable xenograft was obtained by grafting a breast cancer sample into nude mice. The biological and genetic profiles of the xenograft were compared with that of the patient's tumour using histology, immunohistochemistry (IHC), BRCA2 sequencing, comparative genomic hybridisation (CGH), and qRT-PCR. Tumour response to standard chemotherapies was evaluated. RESULTS: Histological profile identified the tumour as a basal-like triple-negative breast cancer. Targeted BRCA2 DNA sequencing of the xenograft showed the presence of the mutation previously identified in the carrier. Comparative genomic hybridisation array profiles of the primary tumour and the xenograft revealed a high number of similar genetic alterations. The therapeutic assessment of the xenograft showed sensitivity to anthracyclin-based chemotherapy and resistance to docetaxel. The xenograft was also highly sensitive to radiotherapy and cisplatin-based treatments. CONCLUSIONS: This study describes a new human breast cancer xenograft obtained from a BRCA2-mutated patient. This xenograft provides a new model for the pre-clinical drug development and for the exploration of the drug response biological basis.

Combination of PI3K and MEK inhibitors yields durable remission in PDX models of PIK3CA-mutated metaplastic breast cancers.

BACKGROUND: Metaplastic breast cancer (MBC) is a rare form of breast cancer characterized by an aggressive clinical presentation, with a poor response to standard chemotherapy. MBCs are typically triple-negative breast cancers (TNBCs), frequently with alterations to genes of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways. The objective of this study was to determine the response to PI3K and MAPK pathway inhibitors in patient-derived xenografts (PDXs) of MBCs with targetable alterations. METHODS: We compared survival between triple-negative MBCs and other histological subtypes, in a clinical cohort of 323 TNBC patients. PDX models were established from primary breast tumors classified as MBC. PI3K-AKT-mTOR and RTK-MAPK pathway alterations were detected by targeted next-generation sequencing (NGS) and analyses of copy number alterations. Activation of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways was analyzed with reverse-phase protein arrays (RPPA). PDXs carrying an activating mutation of PIK3CA and genomic changes to the RTK-MAPK signaling pathways were treated with a combination consisting of a PI3K inhibitor and a MEK inhibitor. RESULTS: In our clinical cohort, the patients with MBC had a worse prognosis than those with other histological subtypes. We established nine metaplastic TNBC PDXs. Three had a pathogenic mutation of PIK3CA and additional alterations to genes associated with RTK-MAPK signaling. The MBC PDXs expressed typical EMT and stem cell genes and were of the mesenchymal or mesenchymal stem-like TNBC subtypes. On histological analysis, MBC PDXs presented squamous or chondroid differentiation. RPPA analysis showed activation of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways. In vivo, the combination of PI3K and MAPK inhibitors displayed marked antitumor activity in PDXs carrying genomic alterations of PIK3CA, AKT1, BRAF, and FGFR4. CONCLUSION: The treatment of metaplastic breast cancer PDXs by activation of the PI3K-AKT-mTOR and RTK-MAPK pathways at the genomic and protein levels with a combination of PI3K and MEK inhibitors resulted in tumor regression in mutated models and may therefore be of interest for therapeutic purposes.

CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancers xenografts.

CD44 is a marker of tumour-initiating cells and is upregulated in invasive breast carcinoma; however, its role in the cancer progression is unknown. Here, we show that antibody-mediated CD44-targeting in human breast cancer xenografts (HBCx) significantly reduces tumour growth and that this effect is associated to induction of growth-inhibiting factors. Moreover, treatment with this antibody prevents tumour relapse after chemotherapy-induced remission in a basal-like HBCx.

Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.

BACKGROUND: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. METHODS: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. RESULTS: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. CONCLUSION: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

CXCR4 inhibitors could benefit to HER2 but not to triple-negative breast cancer patients.

The CXCR4 receptor and its ligand CXCL12 (also named stromal cell-derived factor 1, SDF1) have a critical role in chemotaxis and homing, key steps in cancer metastasis. Although myofibroblasts expressing CXCL12 are associated with the presence of axillary metastases in HER2 breast cancers (BC), the therapeutic interest of targeting CXCR4/CXCL12 axis in the different BC subtypes remains unclear. Here, we investigate this question by testing antitumor activity of CXCR4 inhibitors in patient-derived xenografts (PDX), which faithfully reproduce human tumor properties. We observed that two CXCR4 inhibitors, AMD3100 and TN14003, efficiently impair tumor growth and metastasis dissemination in both Herceptin-sensitive and Herceptin-resistant HER2 BC. Conversely, blocking CXCR4/CXCL12 pathway in triple-negative (TN) BC does not reduce tumor growth, and can even increase metastatic spread. Moreover, although CXCR4 inhibitors significantly reduce myofibroblast content in all BC subtypes, they decrease angiogenesis only in HER2 BC. Thus, our findings suggest that targeting CXCR4 could provide some therapeutic interest for HER2 BC patients, whereas it has no impact or could even be detrimental for TN BC patients.

The molecular mechanisms underlying the ERα-36-mediated signaling in breast cancer.

Alterations in estrogen-mediated cellular signaling have largely been implicated in the pathogenesis of breast cancer. Here, we investigated the signaling regulation of a splice variant of the estrogen receptor, namely estrogen receptor (ERα-36), associated with a poor prognosis in breast cancers. Coupling in vitro and in vivo approaches we determined the precise sequential molecular events of a new estrogen signaling network in an ERα-negative cell line and in an original patient-derived xenograft. After estrogen treatment, ERα-36 rapidly associates with Src at the level of the plasma membrane, initiating downstream cascades, including MEK1/ERK activation and paxillin phosphorylation on S126, which in turn triggers a higher expression of cyclin D1. Of note, the direct binding of ERα-36 to ERK2 prevents its dephosphorylation by MKP3 and enhances the downstream signaling. These findings improve our understanding of the regulation of non-genomic estrogen signaling and open new avenues for personalized therapeutic approaches targeting Src or MEK in ERα-36-positive patients.

A Ku80 fragment with dominant negative activity imparts a radiosensitive phenotype to CHO-K1 cells.

DNA non-homologous end joining, the major mechanism for the repair of DNA double-strands breaks (DSB) in mammalian cells requires the DNA-dependent protein kinase (DNA-PK), a complex composed of a large catalytic subunit of 460 kDa (DNA-PKcs) and the heterodimer Ku70-Ku80 that binds to double-stranded DNA ends. Mutations in any of the three subunits of DNA-PK lead to extreme radiosensitivity and DSB repair deficiency. Here we show that the 283 C-terminal amino acids of Ku80 introduced into the Chinese hamster ovary cell line CHO-K1 have a dominant negative effect. Expression of Ku(449-732) in CHO cells was verified by northern blot analysis and resulted in decreased Ku-dependent DNA end-binding activity, a diminished capacity to repair DSBs as determined by pulsed field gel electrophoresis and decreased radioresistance determined by clonogenic survival. The stable modifications observed at the molecular and cellular level suggest that this fragment of Ku80 confers a dominant negative effect providing an important mechanism to sensitise radioresistant cells.

Transfusion of stored red blood cells in critically ill trauma patients: a retrospective study.

OBJECTIVE: The many published studies on the effects of the transfusion of stored red blood cells on clinical outcomes yielded discordant results. Therefore, we chose to study patients with severe trauma. The clinical outcomes considered included in-hospital mortality, the occurrence of sepsis, length of stay in intensive care unit and in hospital, and days of mechanical ventilation. PATIENTS AND METHODS: We selected all patients with traumatic injury, who received at least 2 red cell units in the first day of admission. Patients were divided into two groups: those who had received fresh red cells only (fresh group) and those who had received at least one "old" red cell unit (old group). The red cells were considered fresh if they had been stored <14 days. RESULTS: The fresh and old groups included 376 and 321 patients, respectively. Baseline demographic and clinical characteristics were comparable between the groups. However, old group received more red cell and plasma units during whole hospital stay (red cells: 11 ± 7 vs 6 ± 4, p < 0.001; plasma: 7 [0-9] vs 3 [0-6]). Among outcomes, only length of stay in intensive care unit (old vs fresh: 18 ± 9 vs 12 ± 8 days, p < 0.001) and in hospital (77 ± 35 vs 45 ± 30 days, p < 0.001) differed significantly between groups. The association remained statistically significant in a multivariate analysis including known confounding factors. CONCLUSIONS: Patients with major trauma transfused with old (≥14 days) red cells had a longer length of stay in intensive care unit and in hospital, without any difference in mortality, occurence of sepsis or days of mechanical ventilation.

A methodological approach for determination of maximal inspiratory pressure in patients undergoing invasive mechanical ventilation.

BACKGROUND: Maximal inspiratory pressure (MIP) can help to evaluate inspiratory muscle strength. However its determination in ventilated patients is cumbersome and needs special equipment. We hypothesized that MIP could be obtained by using the expiratory hold knob of the ventilator. The aim of this study was to verify whether: 1) the end expiratory occlusion technique can be used for MIP determination; and 2) if this technique provides different results compared to those obtained by the traditional method of MIP calculation. METHODS: We studied 23 consecutive patients undergoing mechanical ventilation for acute respiratory failure. The MIP was determined by two different methods, both based on occluding the airway for 20 seconds. This occlusion was obtained either by pressing the expiratory hold knob of the ventilator; or by detaching the patient from the ventilator circuit and using a noiseless pneumatic shutter placed on the inspiratory line of a two-way valve that allows expiration but prevents inspiration. RESULTS: The average values of MIP obtained by using either the hold knob of the ventilator or the noiseless pneumatic shutter were -46±14 cmH2O and -56±13 cmH2O, respectively. The linear regression analysis showed a significant correlation between MIPVent and MIPOcc (r2=0.95), although the Bland- Altman analysis revealed that they are not clinically comparable. CONCLUSION: MIP can be easily determined at the bedside by pressing the expiratory hold knob of ventilator. However, MIPVent and MIPOcc are different in terms of absolute value probably because they were determined at diverse lung volume.

Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts.

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitivity and DSB repair after transfection of a Ku86-antisense in a human fibroblast cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were transfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The main endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kinase activity, clonogenic survival, and DSB repair kinetics. After transfection of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-binding activity, and DNA protein kinase activity were observed in the uncloned cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a surviving fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB repair and in the radiosensitivity of human cells. This investigation strongly suggests that Ku86 could constitute an appealing target for combining gene therapy and radiation therapy.

Comparison of diagnostic accuracy of sublingual nitroglycerin test and low-dose isoproterenol test in patients with unexplained syncope.

Despite the widespread use of head-up tilt testing as a means for diagnosing vasovagal syncope, standardization of test methodology remains a controversial issue. The aim of this study was to compare the diagnostic value of head-up tilt testing potentiated with sublingual nitroglycerin with that of head-up tilt testing potentiated with low-dose isoproterenol. For this purpose, 71 patients with unexplained syncope (mean age 43 years) and 30 asymptomatic controls were studied. All underwent the sublingual nitroglycerin and low-dose isoproterenol tests on separate days in a randomized fashion. The protocol of the 2 tests was similar and consisted of 2 phases. Initially, subjects were tilted at 60 degrees for 20 minutes without medication; then, if syncope did not occur, patients and controls received sublingual nitroglycerin (300 microg) or low-dose intravenous isoproterenol (mean infusion rate 1.3 +/- 0.5 microg/min) and continued to be tilted at 60 degrees for a further 20 minutes. During the sublingual nitroglycerin test, a positive response (syncope associated with sudden hypotension and bradycardia) occurred in 35 patients (49%), a negative response in 36 (51%), and drug intolerance in none (0%). During the low-dose isoproterenol test, these percentages were 41%, 59%, and 6%, respectively. A concordant response was observed in 53 cases (75%). Among controls, 3 subjects (10%) had a positive response to the sublingual nitroglycerin test and 4 (13%) to the low-dose isoproterenol test. It is concluded that sublingual nitroglycerin and low-dose isoproterenol are equivalent tests for evaluating patients with unexplained syncope. The sublingual nitroglycerin test, however, is simpler, better tolerated, and safer than the low-dose isoproterenol test and, thus, more suitable for routine clinical use.

Ventricular tachyarrhythmias in Prinzmetal's variant angina: clinical significance and relation to the degree and time course of S-T segment elevation.

Fifty-six patients with active Prinzmetal's variant angina were studied to determine the incidence and clinical significance of ventricular tachyarrhythmias and the correlation between arrhythmias and degree and time course of S-T segment changes during the ischemic attacks. Twenty-nine patients (Group I) had no ventricular arrhythmias in any of the 1,083 recorded episodes, while 27 patients (Group II) developed arrhythmias in 18% of the attacks. No significant differences in clinical, electrocardiographic, angiographic, or hemodynamic findings could be found between the 2 groups. In 23 of the 27 Group II patients, ventricular arrhythmias developed during maximal S-T segment elevation (occlusion arrhythmias), while in 10 they occurred during resolution of S-T segment changes (reperfusion arrhythmias); 6 of the latter patients also had occlusion arrhythmias. Eight of the 23 patients with occlusion arrhythmias and 6 of the 10 with reperfusion arrhythmias had ventricular fibrillation or ventricular tachycardia. Maximal S-T segment elevation was significantly greater (p less than 0.001) in patients with occlusion arrhythmias than in those without arrhythmias. The episodes with reperfusion arrhythmias were significantly longer (p less than 0.001) and showed a significantly greater S-T segment elevation (p less than 0.001) than those without arrhythmias in Group I patients. This study shows that significant ventricular tachyarrhythmias develop during ischemic attacks in about 50% of patients with active variant angina; clinical and angiographic features are not useful in distinguishing patients with arrhythmias from the others. Our findings suggest that in variant angina ventricular arrhythmias may be due to the effects of both coronary artery occlusion and reperfusion; both types of arrhythmias are correlated with the severity of ischemia, as measured by the degree of S-T segment elevation. Reperfusion arrhythmias also appear to be correlated with the duration of ischemia.

Responsiveness to intravenous administration of salbutamol in chronic obstructive pulmonary disease patients with acute respiratory failure.

OBJECTIVE: In chronic obstructive pulmonary disease (COPD) patients with acute respiratory failure (ARF), bronchodilating agents administered by inhalation have, in general, little effect on dynamic hyperinflation and concurrent static intrinsic positive end-expiratory pressure (PEEPi,st). Since in COPD the severely obstructed segments of the lung may not be reached by inhaled medication, we reasoned that drug efficiency may be enhanced by intravenous administration of the agent. DESIGN: Physiological study. SETTING: Two four-bed surgical-medical ICUs of a university hospital. PATIENTS: Fourteen COPD patients were studied within 36 h from the onset of ARF. MEASUREMENTS AND RESULTS: Static compliance (Cst,rs), minimal (Rmin,rs) and additional (DeltaRrs) resistance of the respiratory system, and PEEPi,st were measured before and after intravenous administration of salbutamol. All patients had limitation of air flow before and after salbutamol administration. On average, after salbutamol there was a small, though significant, decrease in Rmin,rs (-9%), DeltaRrs (-12%) and PEEPi,st (-8%). CONCLUSION: The changes in resistance and PEEPi,st after intravenous administration of salbutamol were too small to be of clinical significance.

Coexistence of mitochondrial DNA and beta myosin heavy chain mutations in hypertrophic cardiomyopathy with late congestive heart failure.

OBJECTIVE: To investigate the possible coexistence of mitochondrial DNA (mtDNA) mutations in patients with beta myosin heavy chain (beta MHC) linked hypertrophic cardiomyopathy (HCM) who develop congestive heart failure. DESIGN: Molecular analysis of beta MHC and mtDNA gene defects in patients with HCM. SETTING: Cardiovascular molecular diagnostic and heart transplantation reference centre in north Italy. PATIENTS: Four patients with HCM who underwent heart transplantation for end stage heart failure, and after pedigree analysis of 60 relatives, eight additional affected patients and 27 unaffected relatives. A total of 111 unrelated healthy adult volunteers served as controls. Disease controls included an additional 27 patients with HCM and 102 with dilated cardiomyopathy. INTERVENTION: Molecular analysis of DNA from myocardial and skeletal muscle tissue and from peripheral blood specimens. MAIN OUTCOME MEASURES: Screening for mutations in beta MHC (exons 3-23) and mtDNA tRNA (n = 22) genes with denaturing gradient gel electrophoresis or single strand conformational polymorphism followed by automated DNA sequencing. RESULTS: One proband (kindred A) (plus seven affected relatives) had arginine 249 glutamine (Arg249Gln) beta MHC and heteroplasmic mtDNA tRNAIle A4300G mutations. Another unrelated patient (kindred B) with sporadic HCM had identical mutations. The remaining two patients (kindred C), a mother and son, had a novel beta MHC mutation (lysine 450 glutamic acid) (Lys450Glu) and a heteroplasmic missense (T9957C, phenylalanine (Phe)-->leucine (Leu)) mtDNA mutation in subunit III of the cytochrome C oxidase gene. The amount of mutant mtDNA was higher in the myocardium than in skeletal muscle or peripheral blood and in affected patients than in asymptomatic relatives. Mutations were absent in the controls. Pathological and biochemical characteristics of patients with mutations Arg249Gln plus A4300G (kindreds A and B) were identical, but different from those of the two patients with Lys450Glu plus T9957C(Phe-->Leu) mutations (kindred C). Cytochrome C oxidase activity and histoenzymatic staining were severely decreased in the two patients in kindreds A and B, but were unaffected in the two in kindred C. CONCLUSIONS: beta MHC gene and mtDNA mutations may coexist in patients with HCM and end stage congestive heart failure. Although beta MHC gene mutations seem to be the true determinants of HCM, both mtDNA mutations in these patients have known prerequisites for pathogenicity. Coexistence of other genetic abnormalities in beta MHC linked HCM, such as mtDNA mutations, may contribute to variable phenotypic expression and explain the heterogeneous behaviour of HCM.