Confocal Laser Scanning Microscopy Approach to Investigate Plant-Fungal Interactions.

Plants interact with a broad range of microorganisms, such as bacteria and fungi. In plant roots, complex microbial communities participate in plant nutrition and development as well as in the protection against stresses. The establishment of the root microbiota is a dynamic process in space and time regulated by abiotic (e.g., edaphic, climate, etc.) and biotic factors (e.g., host genotype, root exudates, etc.). In the last 20 years, the development of metabarcoding surveys, based on high-throughput next-generation sequencing methods, identified the main drivers of microbial community structuration. However, identification of plant-associated microbes by sequencing should be complemented by imaging techniques to provide information on the micrometric spatial organization and its impact on plant-fungal and fungal-fungal interactions. Laser scanning confocal microscopy can provide both types of information and is now used to investigate communities of endophytic, endomycorrhizal, and ectomycorrhizal fungi. In this chapter, we present a protocol enabling the detection of fungal individuals and communities associated to the plant root system.

Colonization of Naive Roots from Populus tremula × alba Involves Successive Waves of Fungi and Bacteria with Different Trophic Abilities.

Through their roots, trees interact with a highly complex community of microorganisms belonging to various trophic guilds and contributing to tree nutrition, development, and protection against stresses. Tree roots select for specific microbial species from the bulk soil communities. The root microbiome formation is a dynamic process, but little is known on how the different microorganisms colonize the roots and how the selection occurs. To decipher whether the final composition of the root microbiome is the product of several waves of colonization by different guilds of microorganisms, we planted sterile rooted cuttings of gray poplar obtained from plantlets propagated in axenic conditions in natural poplar stand soil. We analyzed the root microbiome at different time points between 2 and 50 days of culture by combining high-throughput Illumina MiSeq sequencing of the fungal ribosomal DNA internal transcribed spacer and bacterial 16S rRNA amplicons with confocal laser scanning microscopy observations. The microbial colonization of poplar roots took place in three stages, but bacteria and fungi had different dynamics. Root bacterial communities were clearly different from those in the soil after 2 days of culture. In contrast, if fungi were also already colonizing roots after 2 days, the initial communities were very close to that in the soil and were dominated by saprotrophs. They were slowly replaced by endophytes and ectomycorhizal fungi. The replacement of the most abundant fungal and bacterial community members observed in poplar roots over time suggest potential competition effect between microorganisms and/or a selection by the host.IMPORTANCE The tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development, and protection against different types of stress. They mainly originate from the bulk soil and colonize the root system, which provides a unique nutrient-rich environment for a diverse assemblage of microbial communities. In order to better understand how the tree root microbiome is shaped over time, we observed the composition of root-associated microbial communities of naive plantlets of poplar transferred in natural soil. The composition of the final root microbiome relies on a series of colonization stages characterized by the dominance of different fungal guilds and bacterial community members over time. Our observations suggest an early stabilization of bacterial communities, whereas fungal communities are established following a more gradual pattern.

[The combined radiochemotherapy of inoperable esophageal neoplasms: a feasibility study]. / Radio-chemioterapia combinata delle neoplasie esofagee inoperabili: studio di fattibilità.

January, 1994, through January, 1995, eighteen patients (17 men; median age: 59.9, range: 32-73) with biopsy-proved squamous cell carcinoma (n = 15), adenocarcinoma (n = 2) or undifferentiated carcinoma (n = 1) of the esophagus were treated with concurrent chemo-radiotherapy. All patients had inoperable lesions for unresectable disease (11 patients) or concomitant illness (7 patients); median Karnofsky score was 70 (range: 60-80). According to the 1988 American Joint Committee on Cancer Staging system, one patient was graded as Stage IIA (T2N0 + oropharyngeal cancer T4N1), two Stage IIB (T2N1), twelve Stage III (8 T3N1, 1 T4N0, 3 T4N1) and three Stage IV (2 T3N0M1, 1 T4N0M1). Treatment consisted of two courses of chemotherapy by cisplatin (75 mg/m2 i.v. on days 1 and 29) and 5-FU (1000 mg/m2/24 hours by continuous infusion from days 1 to 4 and from days 29 to 32) along with one course of concomitant radiotherapy at 45 Gy (1.8 Gy per fraction, one fraction per day and 5 fractions a week). After 15-30 days, the patients were treated with a boost dose of 7 Gy by high-dose-rate intraluminal brachytherapy. All patients are assessable for toxicity and seventeen for response. The combined treatment was generally well tolerated, with only one case of WHO grade III toxicity (thrombocytopenia). Eight of the eighteen patients had a complete response (47%); four a partial response (24%); four a minimal response (24%) and one showed stable disease (5%). Only one patient developed local progression, and four distant metastases. All the eight patients with CR are alive without local recurrence (two distant metastases) with a mean follow-up of 6 months. This treatment regimen provides good local tumor resolution with no major toxicity. The value of this study protocol will be determined by the rate of long-term survivors.

[The results of sandwich adjuvant radiotherapy in 2nd- and 3rd-stage rectal adenocarcinoma. The authors' personal experience]. / Risultati della radioterapia adiuvante a "sandwich" nell'adenocarcinoma del retto negli stadi II e III. Esperienza personale.

From January, 1985, to June, 1993, 125 patients with stages B2-C adenocarcinomas of the rectum were submitted to pre- and postoperative irradiation according to Thomas Jefferson University protocol guidelines. Five hundred cGy were administered as a single preoperative dose 24 hours before surgery using parallel opposed (AP-PA) treatment fields including the whole pelvis. Pathologic samples were classified following the Astler-Coller staging criteria. Forty-seven patients had no postoperative treatment because their disease stage was A, B1 or D, 11 for refused consent and 9 postoperative complications preventing any further therapy. Seventy-eight patients concluded the treatment schedule and are assessable for response. Radiotherapy total dose consisted of 4400-5000 cGy administered over 5-6 weeks: the patients were treated with megavoltage photons (15-MeV photons) and one dose fraction of 2 Gy was delivered daily, 5 days a week, with the "box" or the "three-field" technique. Median follow-up time was 50.2 months from the beginning of treatment for all the patients in our series (range: 18-120 months). Radiation therapy was well tolerated: 5 patients had severe diarrhea and 2 had small bowel obstruction which required surgery. Local recurrences were observed in 13 of 78 patients (16.7%). Overall actuarial survival at 5 years was 66.8%. Our results confirm the efficacy of this treatment, which is in agreement with international literature data. However, no difference was seen relative to the results obtained with postoperative irradiation alone. We conclude that sandwich radiotherapy can be an effective tool for the local control of rectal adenocarcinoma, with acceptable morbidity, even though it fails to prevent metastases.

[RAdio-chemo-surgical combined treatment of non-small cell lung carcinoma N2]. / Trattamento integrato radio-chemio-chirurgico nel carcinoma polmonare non a piccole cellule N2.

As yet, no optimal treatment for stage-IIIA non-small-cell lung cancer (NSCLC) has been established. Particularly, in the patients with stage-IIIA N2 disease, surgical resection for cure is limited to few selected patients. Of late, a number of studies have suggested that such treatment modalities as chemotherapy, radiotherapy and surgery might be combined to improve treatment efficacy. Based on these conclusions, a cooperative study for N2 NSCLC patients was performed. Treatment included continuous CDDP infusion (6 mg/m2/day) and concomitant irradiation. Fifteen patients were examined. After neoadjuvant treatment, 4 patients were found to have unresectable lesions for local disease progression or metastasis. Eleven patients underwent complete resection (73% resectability). Follow-up ranged 6 to 32 months: 6 patients are now free from relapse (respectively at 31, 28, 23, 14, 12 and 3 months) and 1 is alive with adrenal gland metastasis. Overall and disease-free survival rates are 40.6% and 31.5%, respectively. Our preliminary results indicated that this protocol is well tolerated. Resectability was good and tumor sterilization rate was satisfying (complete T and N sterilization in 6 cases, sterilization of either T or N in 3 cases). The patients with non-adenocarcinoma histology exhibited better local control and prognosis than those with histologic diagnosis of adenocarcinoma.

[The use of cisplatin as radiosensitizing agent in advanced tumors of the head and neck. Randomized study]. / Utilizzazione del cisplatino come radiosensibilizzante nei tumori avanzati della regione testa-collo. Studio randomizzato.

This study was aimed at assessing whether c-DDP administration immediately before radiotherapy could increase frequency and duration of objective responses, as well as survival, in patients affected with locally advanced stages of squamous carcinomas of the head and neck. All patients had already undergone two induction cycles according to the CABO schedule. Ninety-six of 108 treated patients could be evaluated. Treatment schedule consisted in: 1) randomized distribution of patients into two groups before induction chemotherapy; 2) two cycles of induction chemotherapy according to the CABO schedule in all patients; 3) radiation therapy: the patients in group A were given 5 mg/mq of i.v. cisplatin, 30-60 minutes before each session. The results from the two groups were compared and no significant differences were observed regarding objective response (82.5% in group A vs. 86% in group B), response duration and overall survival rates. Even though toxicity was higher in the patients in group A, therapeutic protocol never needed be modified.

[The role of radiotherapy in the treatment of vulvar tumors]. / II ruolo della radioterapia nel trattamento del tumore della vulva.

Carcinoma of the vulva is relatively rare, making up 3% to 4% of all primary genital cancer. It is a disease of the elderly. The Authors treated from 1976 to 1987 twenty-one patients with locally advanced squamous vulvar cancer (6 T2; 12 T3; 3 T4). Nine of these patients were submitted to radiotherapy alone; the others were treated with a combination of surgery and postoperative radiation. Two and five years disease-free survival rate was 33% and 19%. The rate of radiotherapy sequelae has been low, because the patients were treated with the concentional 200 cGy per day or similar fractionation schemes. Disease-free survival rate was better in the subgroup of patients who underwent to combined surgery and post-operative radiation therapy. These results are discussed in comparison with others series in literature.

[The use of electrons in oncologic radiotherapy: our experience]. / L'uso degli elettroni nella radioterapia oncologica: nostra esperienza.

The authors describe the interaction of electrons with tissues, the characteristics of depth isodose curves with sharp dose fall-off. These characteristics reduce the utilization of electron therapy only for tumors situated some 5 cm depth below the skin surface and with regular surface. The authors report their experience from 1978 in the treatment of vulvar carcinoma, chest-wall recurrences from breast carcinoma, and cutaneous lymphomas. All these neoplastic diseases were treated with electron beam of adequate energy. Initially betatron was used, successively a linear accelerator (Siemens) about for two years. Results obtained in the treatment of vulvar carcinoma are reported. Two and five years disease free survival rates was respectively 33% and 19% (it is remarked the importance of prophylactic treatment on inguinal lymphnodes). Local control observed in 44 patients with cutaneous lymphomas was 85% until three years; a greater number of recurrences was observed in centrocytic-centroblastic lymphomas. Moreover, local control of breast recurrences was 65% with 5 years survival rate of 22% (34% in patients with only one skin recurrence). Only 22% of the further local failures appear within the treatment field while 78% appear out of field.

Appearance, site of occurrence, and physical and clinical characteristics of oral carcinoma in Torino, Italy.

The appearance, site of occurrence, and selected physical and clinical characteristics are reported for 102 symptomatic carcinomas diagnosed during a 30-month period in Torino, Italy. Erythroplasia was a more significant visual component than leukoplakia, confirming the results of a previous study on asymptomatic cancer in a US population. Floor of the mouth, oral tongue, and soft palate complex accounted for 75% of all sites and 84% of sites if posterior pillar is excluded. Seventeen percent of the lesions were T1, 60% T2, and 23% larger than T2. Size of the lesion was associated with anterior or posterior position and the mobility of the structure, as well as with ulceration, bleeding, and lymphadenopathy (55% of cancers had no nodal involvement). Tumefaction, burning, and pain were the most frequently reported symptoms. About 50% of patients experienced a time lapse between onset of symptoms and final diagnosis of less than 3 months, and this proportion was higher among patients with T1 cancers.

[Preoperative endocavitary curietherapy of stage Ib-IIa-IIb cervical carcinoma. Personal observations]. / Curieterapia endocavitaria pre-operatoria nel carcinoma della cervice uterina agli stadi Ib-IIa-IIb. Osservazioni personali.

From 1980 through 1984, 41 patients with squamous cell cervix carcinoma and 1 with adenosquamous carcinoma were treated with preoperative irradiation. Clinical stages were Ib in 6 patients, IIa in 24, and IIb in 12. At surgery, lymph node metastases were found in 5 cases, and residual tumors in 8. The latter risk patients were given further external radiotherapy after surgery. Overall three-year survival rates for FIGO stage Ib was 100%; 91.6% for stage IIa, and 83% for stage IIb (minimum follow-up: 3 years). Two patients died from locoregional recurrence of the disease 12-24 months after the treatment, and 2 from distant metastases; 5 patients have showed signs of local improvement. Our results seem to point to pelvic lymph node involvement as the major prognostic factor: in fact, 40% only of the patients with involved lymph nodes is alive. Actuarial survival rates show 90.4% of patients to be alive at 5 years. Tolerance to the combined use radiotherapy and surgery was fair: no severe side-effects were observed. Even though our results are encouraging, a randomized study is still recommended to verify the actual value of this treatment versus combined surgery and radiotherapy or radiotherapy alone.

Chest irradiation as an attempt to improve the response after induction chemotherapy in small cell lung carcinoma.

Forty-six patients with small cell lung carcinoma received cyclic chemotherapy with cisplatin-VP 16 and vincristine, doxorubicin, and cyclophosphamide. The responding patients were given prophylactic cranial irradiation. Patients without metastases not achieving a complete response (CR) following induction chemotherapy were given chest irradiation. The response rate was 73.9 per cent. Response was improved by radiation therapy in only 9 per cent of the patients with limited disease. Median survival was 39 weeks, with 2 patients surviving for longer than 24 months. The duration of response and survival in complete and partial responders was similar; absence of radiation therapy in the patients with CR might explain this finding.