The Frequency of Germline BRCA and Non-BRCA HR-Gene-Variants in a Cohort of Pancreatic Cancer Patients.

Germline DNA alterations affecting homologous recombination pathway genes have been associated with pancreatic cancer (PC) risk. BRCA2 is the most studied gene and affects the management of PC patients and their families. Even though recent reports have suggested a similar role of germline ATM pathogenic variants (PV) in familial PC, there is still a disagreement between experts on how it could affect patient management given the lack of proper PC risk estimates. We retrospectively analyzed the germline data of 257 PC patients among whom nearly 50% were sporadic cases. We showed similar frequencies of BRCA2 (4.9%) and ATM (4.4%) PV or likely pathogenic variants, which were not related to familial history. Based on our findings and that of the literature, we suggest including ATM gene among the panel of genes analyzed in PC patients pending the publication of prospective studies.

Emergence of multidrug-resistant bacteria isolated from surgical site infection in dogs and cats / [Emergência de bactérias multirresistentes isoladas da infecção no sítio cirúrgico em cães e gatos]

Surgical site infections (SSIs) and antimicrobial resistance among pathogens causing SSI are a growing concern in veterinary hospitals. One major reason, the widespread use of antimicrobials, has led to increased incidence of SSIs. This study identified bacteria and resistance profiles to antimicrobials in the SSI cases diagnosed at the Surgical Clinic of Small Animals in the Veterinary Hospital, Federal University of Viçosa, Brazil. The main genus identified was Staphylococcus, followed by Escherichia, Enterococcus, Bacillus, Shigella, Citrobacter, Proteus, Morganella, Serratia, Enterobacter, Pseudomonas and Klebsiella were also found, but in small number. The results indicated the predominance of Gram-negative bacteria among the collected samples. Most of isolates identified were resistant to more than one of the following antimicrobials: ampicillin, tetracycline, enrofloxacin, amoxicillin/clavulanic acid and cephalotin. Of the 17 Staphylococcus sp. isolates, two (11.8%) were methicillin-resistant Staphylococcus aureus (MRSA) and 11 (64.7%) of them were methicillin-resistant Staphylococcus pseudintermedius (MRSP). There were bacterial genera identified with resistance to all tested antimicrobials in different proportions. This should alert veterinary hospitals to the emergence of multidrug-resistant bacteria and to the requirement for the revision of surgical protocols with regard to antimicrobial prophylaxis and therapy.(AU)

As infecções em sítio cirúrgico (ISCs) e a resistência bacteriana entre os patógenos relacionados constituem uma preocupação crescente nos hospitais veterinários. O aumento na incidência de ISCs possui forte relação com o uso amplo e disseminado de antibióticos. O presente estudo identificou bactérias e perfis de resistência a antibióticos nos casos de ISCs diagnosticados na Clínica Cirúrgica de Pequenos Animais do Hospital Veterinário da Universidade Federal de Viçosa, Brasil. O principal gênero identificado foi Staphylococcus, seguido pelos gêneros Escherichia, Enterococcus, Bacillus, Shigella, Citrobacter, Proteus, Morganella, Serratia, Enterobacter, Pseudomonas e Klebsiella, porém, em menor quantidade. Os resultados demonstraram a predominância de bactérias Gram-negativas entre as amostras coletadas. A maioria dos isolados identificados eram resistentes a um ou a mais de um dos seguintes antibióticos: ampicilina, tetraciclina, enrofloxacina, amoxicilina/ácido clavulânico e cefalotina. Entre os 17 isolados de Staphylococcus sp., dois (11,8%) eram Staphylococcus aureus resistentes à meticilina (SARM) e 11 (64,7%) eram Staphylococcus pseudintermedius resistentes à meticilina (SPRM). Houve identificação de gêneros bacterianos com diferentes proporções de resistência para todos os antibióticos avaliados. Esses achados devem alertar os hospitais veterinários para a emergência de bactérias multirresistentes e para a necessidade de revisar a profilaxia e a terapia antimicrobiana referente aos protocolos cirúrgicos.(AU)

Perfluorooctanoic acid alters progesterone activity in human endometrial cells and induces reproductive alterations in young women.

Female fecundity is finely regulated by hormonal signaling, representing a potential target for endocrine-disrupting chemicals. Among the chemicals of most concern are the perfluoroalkyl substances (PFAS), widely used in consumer goods, that are associated with adverse effects on reproductive health. In this context, the endometrium clearly represents an important fertility determining factor. The aim of this study was to investigate PFAS interference on hormonal endometrial regulation. This study was performed within a screening protocol to evaluate reproductive health in high schools. We studied a cohort of 146 exposed females aged 18-21 from the Veneto region in Italy, one of the four areas worldwide heavily polluted with PFAS, and 1080 non-exposed controls. In experiments on Ishikawa cells included UV-Vis spectroscopy, microarray analysis and qPCR. We report a significant dysregulation of the genetic cascade leading to embryo implantation and endometrial receptivity. The most differentially-expressed genes upon PFOA coincubation were ITGB8, KLF5, WNT11, SULT1E1, ALPPL2 and G0S2 (all p < 0.01). By qPCR, we confirmed an antagonistic effect of PFOA on all these genes, which was reversed at higher progesterone levels. Molecular interference of PFOA on progesterone was confirmed by an increase in the intensity of absorption spectra at 250 nm in a dose-dependent manner, but not in the presence of ß-estradiol. Age at menarche (+164 days, p = 0.006) and the frequency of girls with irregular periods (29.5% vs 21.5%, p = 0.022) were significantly higher in the exposed group. Our results are indicative of endocrine-disrupting activity of PFAS on progesterone-mediated endometrial function.

Germline mutation p.N363K in POLE is associated with an increased risk of colorectal cancer and giant cell glioblastoma.

Germline mutations of the POLE gene are responsible for polymerase proofreading-associated polyposis syndrome (PPAP). These mutations were hypothesised to predispose to extra-gastrointestinal tumours (ovary, endometrium, brain), but this association has not been confirmed so far. We report a family with an autosomal dominant inheritance of PPAP due to a c.1089C>A; p.Asn363Lys mutation in the proofreading exonuclease domain of POLE. Ten patients presenting a history of colorectal tumours and three patients with polyposis are indexed in this family. Three carriers (including siblings and a distant cousin at 30, 45 and 52 respectively) and another member (at 37 not tested) presented glioblastoma. This is the second family reported to carry this mutation. Among the four glioblastomas in the family that we report, both show similar pathology: giant cell glioblastoma. These cases suggest that the c.1089C>A germline POLE mutation may confer an increased risk of brain cancer [incidence 17.4% (4/23) in mutation carriers combining the two families]. More observations are needed to support this hypothesis. It seems that not all mutations of POLE are equally associated with extra-gastrointestinal tumours. Although carriers of a mutation responsible for PPAP should benefit from screening for colorectal and uterine cancer, due to the rapid evolution of glioblastoma the value of neurological follow-up and brain imaging screening remains questionable. Nevertheless, considering the limitations of standard therapy for glioblastoma, mutation status could be useful for targeting therapy. The biological mechanism linking POLE mutation to glioblastoma remains to be determined.

Standardization and comparison of nonautomated assays to measure the collagen binding activity of von Willebrand factor.

INTRODUCTION: Diagnosis of von Willebrand disease (VWD) is difficult due to the heterogenic phenotype of patients and to the complex tests that are required for an adequate investigation. The collagen binding assay (VWF:CB) reveals the adhesion capacity of von Willebrand factor (VWF) to collagen and can be useful to reduce the misleading diagnosis of VWD. This study aimed the standardization of 2 nonautomated VWF:CB assays based on ELISA and flow cytometry. METHODS: Plasma samples from 87 patients previously diagnosed with VWD and 22 healthy controls were analyzed. Measurement of the VWF-collagen binding activity was performed using a commercial assay and the 2 tests proposed. VWF:CB/VWF:Ag ratio was calculated for samples and the differentiation between types 1, 2A, and 2M was analyzed. RESULTS: ELISA and flow cytometry tests presented strong correlation with the gold standard test (r2  = .8976 and r2  = .8143, respectively). Tests based on ELISA and flow cytometry presented a bias of +7.2% and -3.6%, respectively. CONCLUSION: The ELISA test demonstrated better performance to detect VWF-collagen binding activity in healthy individuals and VWD patients. This test could differentiate 2A and 2M subtypes using a feasible protocol that can be easily implemented.

Transoral thyroidectomy: a viable surgical option with unprecedented complications-a case series.

PURPOSE: To report the clinical implications of an initial experience with transoral endoscopic thyroidectomy vestibular approach (TOETVA). METHODS: From March to November 2017, five cases of TOETVA were performed. Data reported include patient demographics, indication for surgery, extent of surgery, operative time, the need to convert to cervicotomy, the length of hospital stay and post-operative pain and morbidity. Unconventional complications regarded as specific for TOETVA were reported. The burden of surgery on the patient's quality of life was evaluated using the 36-item short form (SF-36) health survey 1 month after surgery. RESULTS: All patients were females with a mean age of 36 years. They all underwent a right-sided hemithyroidectomy for a solitary thyroid nodule measuring on average 3.5 cm in size. The nodule was reported as Bethesda category II (n = 3), III (n = 1), and IV (n = 1) on fine needle aspiration cytology. The mean operative time was 122 min. Conversion to a transverse cervicotomy was required in one case. None of the patients developed post-operative bleeding, and none experienced vocal fold or mental nerve palsy. Surgical site infection did not occur. All patients developed subcutaneous emphysema that resolved within 12-48 h. All patients reported a long-standing bothersome pulling sensation along the surgical track that resulted in a poor outcome in some scales of the SF-36 survey. Flap perforation occurred in one case. The median VAS score was 3. CONCLUSION: Patients strongly motivated to undergo a novel surgical procedure tailored to their needs and desires should be properly counselled particularly regarding unconventional procedure-related complications.

Less is more: time to expand the indications for minimally invasive video-assisted parathyroidectomy.

PURPOSE: Minimally invasive video-assisted parathyroidectomy (MIVAP) is one of the most widespread targeted parathyroid surgeries for primary hyperparathyroidism (PHP). The aim of this study was to assess its limits and propose an expansion of its indications in the management of parathyroid pathology. METHODS: A retrospective analysis of 77 consecutive patients who underwent MIVAP for PHP between Jan and Oct 2016 was conducted. The adequacy of the procedure and/or the need to convert to a standard cervicotomy was the main outcome of interest. Secondary outcomes of interest included: operative time, postoperative morbidity, postoperative pain assessed by the visual analogue scale (VAS) score, and the length of the surgical incision. RESULTS: There were 64 females and 13 males with a mean age of 51 years. In one patient a concomitant en bloc thyroid lobectomy was required due to features suspicious of parathyroid carcinoma while exploration was required in two other patients. None of these three cases required conversion to standard cervicotomy. The mean operative time, length of incision and VAS score was 31 min, 17 mm and 1.6, respectively. Biochemical cure was achieved in all patients, and no postoperative morbidities were reported. CONCLUSION: MIVAP offers the ability to perform a neck exploration and/or an en bloc thyroid lobectomy without the need to convert to a standard cervicotomy. Therefore, it not only serves as a targeted parathyroid procedure but also a potential alternative to full neck exploration.

Hydroxyapatite, polycaprolactone and alendronate composites for bone regeneration in rabbits' olecranon: histological features / Compósitos de hidroxiapatita, policaprolactona e alendronato na regeneração óssea em olécrano de coelhos: características histológicas

O presente trabalho teve como objetivo avaliar a biocompatibilidade e a osseointegração de compósitos de hidroxiapatita (HA), policaprolactona (PCL) e alendronato (ALN) em defeitos ósseos produzidos no olécrano de coelhos. Trinta e seis coelhos foram distribuídos em quatro grupos, recebendo como tratamento: (1) compósito de HA (49,5%), PCL (49,5%) e ALN (1%); (2) HA (50%) e PCL (50%); (3) PCL (100%); e (4) solução salina 0,9%. As amostras para análise histológica foram coletadas de três animais de cada grupo aos oito, 45 e 90 dias de pós-operatório. No oitavo dia, as bordas do defeito ainda eram identificáveis em todos os grupos. Tecido ósseo novo era formado em contato com o biomaterial apenas nas formulações que incluíam HA. Essas características continuaram evidentes nos outros momentos analisados, embora o defeito estivesse preenchido pelo tecido regenerado. A presença dos biomateriais foi verificada nos três momentos em todos os grupos. Não houve evidências de reação indesejável ao biomaterial. As análises histológicas e histomorfométricas mostraram que os biomateriais são biocompatíveis e aqueles contendo a hidroxiapatita favoreceram a formação óssea no início do processo de regeneração, embora o alendronato não tenha apresentado qualquer efeito.

Hydroxyapatite, polycaprolactone and alendronate composites for bone regeneration in rabbits' olecranon: histological features / Compósitos de hidroxiapatita, policaprolactona e alendronato na regeneração óssea em olécrano de coelhos: características histológicas

O presente trabalho teve como objetivo avaliar a biocompatibilidade e a osseointegração de compósitos de hidroxiapatita (HA), policaprolactona (PCL) e alendronato (ALN) em defeitos ósseos produzidos no olécrano de coelhos. Trinta e seis coelhos foram distribuídos em quatro grupos, recebendo como tratamento: (1) compósito de HA (49,5%), PCL (49,5%) e ALN (1%); (2) HA (50%) e PCL (50%); (3) PCL (100%); e (4) solução salina 0,9%. As amostras para análise histológica foram coletadas de três animais de cada grupo aos oito, 45 e 90 dias de pós-operatório. No oitavo dia, as bordas do defeito ainda eram identificáveis em todos os grupos. Tecido ósseo novo era formado em contato com o biomaterial apenas nas formulações que incluíam HA. Essas características continuaram evidentes nos outros momentos analisados, embora o defeito estivesse preenchido pelo tecido regenerado. A presença dos biomateriais foi verificada nos três momentos em todos os grupos. Não houve evidências de reação indesejável ao biomaterial. As análises histológicas e histomorfométricas mostraram que os biomateriais são biocompatíveis e aqueles contendo a hidroxiapatita favoreceram a formação óssea no início do processo de regeneração, embora o alendronato não tenha apresentado qualquer efeito.(AU)

Description of Methicillin-resistant Staphylococcus pseudintermedius from canine pyoderma in Minas Gerais state, Brazil / Descrição de Staphylococcus pseudintermedius meticilina-resistentes de piodermite canina no estado de Minas Gerais, Brasil

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is of worldwide concern in veterinary medicine. The identification of resistant strains is necessary for proper treatment and the prevention of its propagation among animals. This study aimed to identify S. pseudintermedius isolated from canine pyoderma and evaluate their resistance profiles. Lesions from 25 dogs with pyoderma were sampled. Bacterial isolates were subjected to phenotypic and genotypic analysis for identification of the causative agent. S. pseudintermedius isolates were subjected to SmaI macrorestriction analysis and PFGE for genetic grouping, and PCR to identify the presence of the mecA gene. Their resistance profiles against 12 antimicrobials were also assessed. According to the microbiological analysis, 70 of the 75 isolates obtained were S. pseudintermedius. The isolates presented PFGE patterns, with similarity varying between 84.6 and 100%, and were grouped into 19 clusters. Despite a high frequency of mecA-positive isolates (66 out 70), only 12 presented resistances to oxacillin. Multi-resistance was identified in 29 isolates. The high frequency of MRSP isolated in this study highlights the relevance of identifying resistant strains to lead proper clinical treatment.

Staphylococcus pseudintermedius meticilina-resistente (MRSP) é de preocupação mundial na medicina veterinária. A identificação de cepas resistentes é necessária a um tratamento adequado e à prevenção da sua propagação entre os animais. O objetivo do estudo foi identificar S. pseudintermedius isolados de piodermite canina e avaliar o perfil de resistência. Foram coletadas amostras de lesões de 25 cães com piodermite. Os isolados bacterianos foram submetidos a análises fenotípicas e genotípicas para identificação do agente causador. Isolados de S. pseudintermedius foram submetidos à análise de macrorrestrição por SmaI e PFGE para agrupamento genético e à PCR para identificar a presença do gene mecA. Seu perfil de resistência contra 12 antimicrobianos também foi avaliado. De acordo com a análise microbiológica, 70 dos 75 isolados obtidos foram identificados como S. pseudintermedius. Os isolados apresentaram padrões de PFGE com similaridade variando entre 84.6 e 100% e foram agrupados em 19 grupos genéticos. Apesar da frequência alta de isolados mecA positivos (66 em 70), apenas 12 apresentaram resistência à oxacilina. Multirresistência foi identificada em 29 isolados. A alta frequência de MRSP isolados neste estudo destaca a relevância de se identificarem cepas resistentes para se conduzir um tratamento clínico adequado.

Incidência de infecção do sítio cirúrgico e fatores de risco associados na clínica cirúrgica de pequenos animais / Incidence of surgical site infection and their associated risk factors in the surgical small animal clinic

A infecção do sítio cirúrgico (ISC) apresenta-se como um complicador que possui muitos fatores de risco associados, e a maior parte das informações utilizadas nessa área pela medicina veterinária provém da medicina humana. Objetivou-se com este trabalho determinar a taxa de ISC no HVT-UFV, assim como correlacionar sua incidência com os seguintes fatores de risco: quantidade de pessoas presentes durante a cirurgia, classificação do potencial de contaminação da ferida cirúrgica e utilização de antimicrobiano profilático e ou terapêutico. Para isso, foram colhidas informações sobre a ocorrência de ISC, bem como os fatores de risco citados de 401 prontuários. Esses dados passaram por análise estatística, e foram obtidos os seguintes resultados: 21 pacientes diagnosticados com ISC, o que gerou uma taxa de infecção de 5,24 por cento e associação entre incidência de ISC e cirurgias com risco de contaminação maior que 5 por cento. Os demais fatores de risco avaliados não apresentaram associação, porém, em valores absolutos, foi detectada maior ocorrência de infecção nos procedimentos com maior quantidade de pessoas na sala de cirurgia. Portanto, foi possível observar a importância da obtenção fidedigna de dados e a necessidade do desenvolvimento de um sistema de vigilância epidemiológica voltado para a medicina veterinária...

The surgical site infection (SSI) is a complicating factor that has many risk factors, and most of the information used in this area for veterinary medicine comes from human medicine. The aim of this work was to determine the rate of SSI in the VTH-UFV, and to correlate the incidence of SSI with the following risk factors: surgical wound classification, use of antimicrobial prophylaxis, and therapy and the number of people in the operating room during surgery. For that, information about the occurrence of SSI and the risk factors referred to were collected from 401 medical records. These data underwent statistical analysis and obtained the following results: 21 patients diagnosed with SSI resulting in an infection rate of 5.24 percent and a significant association between the occurrence of SSI and the surgical procedures with contamination risk higher than 5 percent. Other risk factors evaluated showed no association. However, in absolute values, a higher incidence of infection was detected in procedures with a greater number of people in the operating room. Therefore, it was possible to observe the importance of obtaining reliable data and the requirement for developing a surveillance system specific for Veterinary Medicine...

Virtual surgeries in patients with congenital heart disease: a multi-scale modelling test case.

The objective of this work is to perform a virtual planning of surgical repairs in patients with congenital heart diseases--to test the predictive capability of a closed-loop multi-scale model. As a first step, we reproduced the pre-operative state of a specific patient with a univentricular circulation and a bidirectional cavopulmonary anastomosis (BCPA), starting from the patient's clinical data. Namely, by adopting a closed-loop multi-scale approach, the boundary conditions at the inlet and outlet sections of the three-dimensional model were automatically calculated by a lumped parameter network. Successively, we simulated three alternative surgical designs of the total cavopulmonary connection (TCPC). In particular, a T-junction of the venae cavae to the pulmonary arteries (T-TCPC), a design with an offset between the venae cavae (O-TCPC) and a Y-graft design (Y-TCPC) were compared. A multi-scale closed-loop model consisting of a lumped parameter network representing the whole circulation and a patient-specific three-dimensional finite volume model of the BCPA with detailed pulmonary anatomy was built. The three TCPC alternatives were investigated in terms of energetics and haemodynamics. Effects of exercise were also investigated. Results showed that the pre-operative caval flows should not be used as boundary conditions in post-operative simulations owing to changes in the flow waveforms post-operatively. The multi-scale approach is a possible solution to overcome this incongruence. Power losses of the Y-TCPC were lower than all other TCPC models both at rest and under exercise conditions and it distributed the inferior vena cava flow evenly to both lungs. Further work is needed to correlate results from these simulations with clinical outcomes.

A new comprehensive gene expression panel to study tumor micrometastasis in patients with high-risk breast cancer.

The incidence and prognostic relevance of bone marrow (BM) and leukapheresis (PBPC) tumor cell contamination (TCC) in breast cancer patients is still to be circumstantiated. We developed a new comprehensive gene expression panel to study cytokeratins (CK), maspin (MAS) and mammaglobin (MAM) as possible predictors of prognosis. Forty-eight patients undergoing high dose chemotherapy (HDCT) and PBPC support were enrolled and analyzed for TCC on 116 PBPC apheresis and 96 BM obtained at basal conditions. All of the patients were evaluated by reverse transcriptase nested PCR (RT-PCR) for MAM and MAS gene expression and by immunocytochemistry (ICC) and nested RT-PCR to evaluate CK expression. PBPC and BM frequency of CK-positive (+) cells was 12-13% by ICC and 71-73% by RT-PCR respectively. Sixty-seven percent of CK ICC+ samples were MAM RT-PCR+ and 89% of them were MAS RT-PCR+. PBPC and BM frequency of MAM+ cells was 21% and 31% respectively, while for MAS+ cells it was 48% and 52% respectively by RT-PCR. After 71 mo median FU, 16 patients (33%) relapsed and 14 (88%) had BM/PBPC TCC. No marker had an impact on overall survival (OS) but MAS expression on BM and MAM expression on PBPC correlated with a statistically significant improved (p=0.05) and worsened RFS (p=0.06) respectively. These data confirm the activity of MAM as a negative prognostic factor and show for the first time that MAS could work as a tumor suppressor gene even in a clinical setting, since it protects from recurrence.

Thalidomide in multiple myeloma, myelodysplastic syndromes and histiocytosis. Analysis of clinical results and of surrogate angiogenesis markers.

BACKGROUND: Thalidomide, as a single agent, has been recently found to induce a clinical response in one third of refractory or relapsed myeloma patients. Although it has been reported that thalidomide significantly inhibits angiogenesis. it is still unclear whether its clinical effect is mediated, at least in part, by its anti-angiogenic properties. PATIENTS AND METHODS: We evaluated thalidomide as a single agent in myeloma, myelodysplastic syndromes (MDS) and histiocytosis, i.e. hematological diseases characterized by increased angiogenesis, and measured prospectively a number of surrogate angiogenesis markers. RESULTS: Clinical responses were observed in 7 of 17 myeloma and 2 of 5 MDS patients. The histiocytosis patient had a partial response. At the time of the best clinical response, plasma levels of angiogenic growth factors, vascular endothelial growth factor (VEGF) and basic-fibroblast growth factor (b-FGF), were significantly decreased, and flow cytometry indicated a decrease of activated endothelial cells in the bone marrow of responding MDS patients. CONCLUSIONS: These observations confirm thalidomide efficacy in myeloma, suggest a possible use in MDS and histiocytosis and may contribute to the prediction of clinical response and to understanding the mechanism of thalidomide's action.

Endostatin, an antiangiogenic drug, induces tumor stabilization after chemotherapy or anti-CD20 therapy in a NOD/SCID mouse model of human high-grade non-Hodgkin lymphoma.

Both chemotherapy and chimeric anti-CD20 monoclonal antibodies are effective agents against B-cell non-Hodgkin lymphoma (NHL). However, patients achieving remission are at risk of relapse. To evaluate the effect of the antiangiogenic drug endostatin used alone and after the administration of cyclophosphamide (CTX) or the anti-CD20 antibody rituximab, we generated a new model of human NHL by transplanting Namalwa cells intraperitoneally into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. First, we determined the most effective treatment schedule for the drugs assessed. When administered alone, CTX (3 courses of 75 mg/kg of body weight given intraperitoneally), rituximab (3 courses of 25 mg/kg given intraperitoneally), and endostatin (5 courses of 50 microg given subcutaneously) delayed tumor growth, and CTX was the most effective in controlling bulky disease. When given after chemotherapy or immunotherapy, endostatin effectively induced tumor stabilization. When mice given CTX or rituximab on days 3, 5, and 7 after transplantation were randomly assigned to receive endostatin or phosphate-buffered saline on days 15 to 19, tumor growth was prevented in endostatin-treated mice as long as the drug was administered. Furthermore, administration of endostatin on days 25 to 29 after tumor regrowth still induced significant tumor regression, whereas CTX and rituximab were not effective. The specific antiangiogenic action of endostatin was confirmed by in vitro and in vivo studies indicating that the drug inhibited proliferation and induced apoptosis of endothelial (but not of NHL) cells. In conclusion, sequential administration of chemotherapy and endostatin seems promising for treating bulky NHL, and the less toxic sequential administration of rituximab and endostatin is promising for treating limited disease. (

Evaluation of acute toxicities associated with autologous peripheral blood progenitor cell reinfusion in patients undergoing high-dose chemotherapy.

Peripheral blood progenitor cell reinfusion (PBPC) in patients undergoing high-dose chemotherapy (HDC) for poor prognosis malignancies, has been described as causing possible acute gastrointestinal (nausea, vomiting), allergic (oedema, bronchospasm, anaphyl- axis), renal (proteinuria, haematuria) and/or cardiovascular (hypotension, arrhythmia, conduction disturbances, transient ischaemic phenomena) toxicities. To establish the clinical relevance of these observations and the possible relationship with different HDC regimens used, we performed a clinical and instrumental evaluation on 33 patients with advanced breast cancer, non-Hodgkin's lymphoma, Hodgkin's disease, relapsed ovarian cancer, Ewing's sarcoma, extragonadal germinal tumour and small cell lung cancer. They underwent at least one reinfusion each for a total of 51 studied procedures. No patient had a previous history of cardiovascular disease or significant intercurrent illness such as diabetes or liver, renal or neurologic impairment. All patients had totally implanted central venous catheters, through which the transplants had been collected and reinfused without technical consequences. To evaluate cardiovascular function, we continuously monitored 12-lead ECGs, with arterial pressure (AP) measurements every 5 min from the beginning of the procedure to 15 min after the reinfusion ended. We did not observe any significant differences between basal and subsequent steps in AP, heart rate, PQ and QTc time, P wave and QRS complex duration or P wave and QRS electrical axes. No patient showed any ST-T tract pathological abnormality, but one patient developed a transient ectopic atrial rhythm, without any haemodynamic disfunction and with spontaneous reversion to sinus rhythm. No patient complained of symptoms of haemodynamic failure. Gastrointestinal side-effects appeared to be strictly related to speed of reinfusion and to the number of packs reinfused, probably reflecting on the amount of dimethylsulphoxide infused. In one patient a tonic-clonic seizure occurred during a vomiting episode, but no patient developed allergic or renal toxicities. We conclude that PBPC reinfusion, if managed according to the procedure we propose in patients without organic impairment, is a safe procedure not associated either with increased risk of acute arrhythmias or ischaemic or significant systemic acute toxicities. Bone Marrow Transplantation (2000) 25, 173-177.

Idarubicinol myelotoxicity: a comparison of in vitro data with clinical outcome in patients treated with high-dose idarubicin.

We evaluated in vitro the toxicity of idarubicin and its active metabolite idarubicinol on haematopoietic progenitors, using human umbilical cord blood and peripheral blood progenitors to obtain dose-response curves. We treated 16 patients with poor prognosis lymphoma in a phase I-II trial of high-dose idarubicin and melphalan and investigated if idarubicinol persisting in patients' plasma at the time of transplantation (day 0), on day +1 and +2 could result in an inhibition of infused progenitors. Colony inhibition was correlated with pharmacokinetic data and with the time of patients' engraftment. Plasma samples obtained before idarubicin treatment demonstrated a colony-stimulating effect, increasing the cloning efficiency by 72%. The inhibitory activity on colony forming unit granulocyte-macrophage (CFU-GM) of patients' plasma collected on the day of transplantation was lower than expected from dose-response curves (21% measured vs 70% expected). The time to patients' WBC and PLT recovery correlated with the amount of CD34+ cells reinfused and, to a lesser extent, with the colony-inhibiting effect of patients' plasma. The correlation between idarubicinol concentration and CFU-GM inhibition was not significant. These data suggest that plasma drug concentration on the day of stem cell reinfusion may overestimate the toxicity of residual anthracyclines to the transplanted cells.

"Stem cell candidates" purified by liquid culture in the presence of Steel factor, IL-3, and 5FU are strictly stroma-dependent and have myeloid, lymphoid, and megakaryocytic potential.

Berardi et al. (Science 1995; 267:105) reported recently that combined cytokine stimulation and antimetabolite treatment were able to isolate cells with characteristics of hemopoietic stem cells. Bone marrow (BM) low-density cells were cultured for 1 week in the presence of Steel factor (SF), IL-3, and the antimetabolite 5-FU. Following this approach one in 10(5) BM cells were purified. These cells showed no clonogenic potential in soft gel assays but presented a striking myeloid-lymphoid potential as long-term culture-initiating cells (LTC-ICs). We investigated this new "stem cell candidate." following a similar approach we purified one in 55,000/130,000 cells from cord blood and peripheral blood in mobilized cancer patients. These cells displayed no clonogenic potential in methylcellulose assays in the presence of different cytokine combinations and generated very few or no clonogenic progenitors in liquid cultures in the presence of cytokines. When seeded on layers derived from the murine BM stromal cell line M2-10B4, 38-86% of the cells purified using this approach generated multilineage progenitors acting as LTC-lCs. In a different series of studies, after 5-week culture on human BM stromal cell line L87/4 layers, cells were forced to selected lineage differentiation by culture in the presence of low concentrations of SF and high concentrations of lineage-specific cytokines such as Flt3-ligand (myeloid and pre-B cell differentiation), Tpo (megakaryocytic differentiation). IL-7, and IL-2 (pre-B and NK differentiation). After 12-day culture under these conditions, generation of myeloid, pre-B, megakaryocytic, and NK progenitors was assessed by immunohistochemistry, flow cytometry, and mRNA expression of CD7, 14, 19, 41b, S6, and 61. We conclude that this procedure for multilineage progenitor cell purification is simple and effective and could have major implications for gene transfer and stem cell transplantation.