Genomic profiling of late-onset basal cell carcinomas from two brothers with nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant genetic disorder. It is commonly caused by mutations in PTCH1 and chiefly characterized by multiple basal cell carcinomas (BCCs) developing prior to the age of 30 years. In rare cases, NBCCS presents with a late onset of BCC development. OBJECTIVE: To investigate BCC tumorigenesis in two brothers, who showed characteristic features of NBCCS but developed their first BCCs only after the age of 40 years. Two other siblings did not show signs of NBCCS. RESULTS: We obtained blood samples from four siblings and nine BCCs from the two brothers with NBCCS. Whole exome sequencing and RNA sequencing revealed loss of heterozygosity (LOH) of PTCH1 in eight out of nine tumours that consistently involved the same haplotype on chromosome 9. This haplotype contained a germinal splice site mutation in PTCH1 (NM_001083605:exon9:c.763-6C>A). Analysis of germline DNA confirmed segregation of this mutation with the disease. All BCCs harboured additional somatic loss-of-function (LoF) mutations in the remaining PTCH1 allele which are not typically seen in other cases of NBCCS. This suggests a hypomorphic nature of the germinal PTCH1 mutation in this family. Furthermore, all BCCs had a similar tumour mutational burden compared to BCCs of unrelated NBCCS patients while harbouring a higher number of damaging PTCH1 mutations. CONCLUSIONS: Our data suggest that a sequence of three genetic hits leads to the late development of BCCs in two brothers with NBCCS: a hypomorphic germline mutation, followed by somatic LOH and additional mutations that complete PTCH1 inactivation. These genetic events are in line with the late occurrence of the first BCC and with the higher number of damaging PTCH1 mutations compared to usual cases of NBCCS.

Clinical activity after fingolimod cessation: disease reactivation or rebound?

BACKGROUND AND PURPOSE: There is debate as to whether the apparent rebound after fingolimod discontinuation is related to the discontinuation itself or whether it is due to the natural course of highly active multiple sclerosis (MS). Our aim was to survey the prevalence of severe reactivation and rebound after discontinuation of fingolimod in a cohort of Italian patients with MS. METHODS: Patients with relapsing-remitting MS who were treated with fingolimod for at least 6 months and who stopped treatment for reasons that were unrelated to inefficacy were included in the analysis. RESULTS: A total of 100 patients who had discontinued fingolimod were included in the study. Fourteen patients (14%) had a relapse within 3 months after fingolimod discontinuation, and an additional 12 (12%) had a relapse within 6 months. According to this study's criteria, 10 patients (10%) had a severe reactivation. Amongst these patients, five (5%) had a reactivation that was considered to be a rebound. CONCLUSIONS: The present study showed that more than 26% of patients are at risk of having a relapse within 6 months after fingolimod discontinuation. Nevertheless, the risk of severe reactivations and rebound is lower than has been previously described.

Is human fibrin sealant a possible choice for the fixation of laparoscopic inguinal hernia repair? A single center experience and the analysis of the results after 326 TAPP in two years.

BACKGROUND: Groin hernioplasty is most intervention performed in the worldwide. The present study aimed to evaluate a combination between ultralight mesh and fibrin human sealant in the laparoscopic treatment of inguinal hernia. PATIENTS AND METHODS: This retrospective study included consecutive patients who underwent laparoscopic transabdominal preperitoneal hernioplasty (TAPP) from 1st of January 2015 to 31st of December 2016. Demographics, surgical data and postoperative outcomes were entered in an anonymized prospective database. Prospective longterm follow-up carried out in all patients. RESULTS: One hundred eighty-four patients with a median age 57.5 (range 19-84) and median BMI (Body Mass Index) of 28.5 (range 18.5-31.5) were included. A median follow-up of 25 months (median 13-35) was carried out. Five recurrences (1.5%) and two cases of missed lipoma were observed and operated. Chronic pain (CP) was observed in eleven (9%) patients but in 9 patients it decreased spontaneously. CONCLUSIONS: Combination of ultralight mesh and Evicel® in TAPP operation is a safe solution for the treatment of groin hernia. Recurrences and chronic pain are similar to other combination of fibrin sealant and meshes. Moreover action of Evicel® may provide to improve the hemostasis and consequently hematoma of the cord.

Immunomodulation in human and experimental arthritis: including vitamin D, helminths and heat-shock proteins.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that is mainly directed to the joints, affecting the synovial membrane, the cartilage and also the bone. This disease affects 1% to 2% of the world population and is associated with significant morbidity and increased mortality. RA experimental models have allowed a great deal of information to be translated to the corresponding human disease. This review summarizes some of the most relevant findings targeting immunomodulation in arthritis. Some general guidelines to choose an adequate experimental model and also our experience with arthritis are supplied.

Predicting response to neoadjuvant chemotherapy with liquid biopsies and multiparametric MRI in patients with breast cancer.

Accurate prediction of response to neoadjuvant chemotherapy (NAC) can help tailor treatment to individual patients' needs. Little is known about the combination of liquid biopsies and computer extracted features from multiparametric magnetic resonance imaging (MRI) for the prediction of NAC response in breast cancer. Here, we report on a prospective study with the aim to explore the predictive potential of this combination in adjunct to standard clinical and pathological information before, during and after NAC. The study was performed in four Dutch hospitals. Patients without metastases treated with NAC underwent 3 T multiparametric MRI scans before, during and after NAC. Liquid biopsies were obtained before every chemotherapy cycle and before surgery. Prediction models were developed using penalized linear regression to forecast residual cancer burden after NAC and evaluated for pathologic complete response (pCR) using leave-one-out-cross-validation (LOOCV). Sixty-one patients were included. Twenty-three patients (38%) achieved pCR. Most prediction models yielded the highest estimated LOOCV area under the curve (AUC) at the post-treatment timepoint. A clinical-only model including tumor grade, nodal status and receptor subtype yielded an estimated LOOCV AUC for pCR of 0.76, which increased to 0.82 by incorporating post-treatment radiological MRI assessment (i.e., the "clinical-radiological" model). The estimated LOOCV AUC was 0.84 after incorporation of computer-extracted MRI features, and 0.85 when liquid biopsy information was added instead of the radiological MRI assessment. Adding liquid biopsy information to the clinical-radiological resulted in an estimated LOOCV AUC of 0.86. In conclusion, inclusion of liquid biopsy-derived markers in clinical-radiological prediction models may have potential to improve prediction of pCR after NAC in breast cancer.

Bacille Calmette-Guérin/DNAhsp65 prime-boost is protective against diabetes in non-obese diabetic mice but not in the streptozotocin model of type 1 diabetes.

Type I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime-boost strategy involving bacille Calmette-Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non-obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non-immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime-boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases.

Application of computed tomography to assess the effect of egg yolk ratio on body composition in chickens of different genotype and gender at hatch and during the rearing period.

1. Computed tomography was used for the in vivo determination of yolk ratio in 7000 domestic hen eggs, originated from two markedly different genotypes, in order to examine the effect of egg yolk ratio, gender and genotype on the development of hatched chicks. 2. Eggs with extremely low, average and extremely high yolk ratio were chosen for further investigation (n = 350 in each group in both genotypes). After incubating the selected eggs, hatched birds were reared and slaughtered at 11 weeks of age. 3. The yolk ratio of eggs significantly affected the body composition of the chickens at hatching and during rearing to 8-9 weeks of age. 4. Chickens of a genotype selected partly for weight gain had significantly higher live weight from hatching to the end of the rearing period and had better slaughter characteristics than those of the dual purpose TETRA-H genotype. 5. At hatching, only the dry matter content of the body was affected by the gender of the birds. The effect of gender on the live weight and body composition of chickens was evident from the 5th week of age to the time of slaughter. 6. It was concluded that eggs with a low yolk ratio were most beneficial for broiler production, because chickens hatched from these eggs had the highest slaughter weight and muscle index and the lowest fat index for their body mass.

Nationwide analysis of open groin hernia repairs in Italy from 2015 to 2020.

INTRODUCTION: Inguinal hernia repair is one of the most commonly performed operations in general surgery. A total of 130.000 inguinal hernia repairs are performed yearly in Italy, and approximately 20 million inguinal hernias are treated worldwide annually. This report represents the trend analysis in inguinal hernia repair in Italy from a nationwide dataset for the 6-year period from 2015 to 2020. MATERIALS AND METHODS: Based on regional hospital discharge records, all the inguinal hernia repairs performed in public and private hospitals in Italy between 2015 and 2020 were reviewed based on diagnosis and procedure codes. For the aim of this study, data from the AgeNas (The National Agency for Regional Health Services) data source were analyzed. RESULTS: Elective inguinal hernia repairs outnumbered urgent operations over the 6-year study period, ranging from 122,737 operations in 2015 to 65,780 in 2020 as absolute numbers, and from 87.96 to 83.3% of total procedures in 2019 and 2020 respectively, with an annual change ranging from - 66.58%, between 2020 and 2019, to - 2.49%, between 2019 and 2018 (mean = - 18.74%; CI =- 46.7%-9.22%; p < 0.0001). CONCLUSIONS: This large-scale review of groin hernia data from a nationwide Italian dataset provides a unique opportunity to obtain a snapshot of open groin hernia repair activity. More specifically, there is a trend to perform more elective than urgent procedures and there is a steady decrease in the amount of open hernia repairs in favor to laparoscopy.

Laparoscopic approach in emergency for the treatment of acute incarcerated groin hernia: a systematic review and meta-analysis.

PURPOSE: Minimally invasive approach for acute incarcerated groin hernia repair is still debated. To clarify this debate, a literature review was performed. METHODS: Search was performed in PubMed, Embase, Scopus, Web of Science, and Cochrane databases, founding 28,183 articles. RESULTS: Fifteen articles, and 433 patients were included (16 bilateral hernia, range 3-8). Three hundred and eighty-eight (75.3%) and 103 patients (22.9%) underwent transabdominal preperitoneal and totally extraperitoneal repair, respectively, and in 5 patients, the defect was buttressed with broad ligament (1.1%) (not specified in 3 patients). Herniated structures were resected in 48 cases (range 1-9). Intraoperative complications and conversion occurred in 4 (range 0-1) and 10 (range 0-3) patients, respectively. Mean operative time and hospital stay ranged between 50 and 147 min, and 2 and 7 days, respectively. Postoperative complications ranged between 1 and 19. Five studies compared laparoscopic and open approaches (163 and 235 patients). Herniated structures were resected in 19 (11.7%) and 42 cases (17.9%) for laparoscopic and open approach, respectively (p = 0.1191). Intraoperative complications and conversion occurred in one (0.6%) and 5 (2.1%) patients (p = 0.4077), and in two (1.2%) and 19 (8.1%) patients (p = 0.0023), in case of laparoscopic or open approach, respectively. Mean operative time and hospital stay were 94.4 ± 40.2 and 102.8 ± 43.7 min, and 4.8 ± 2.2 and 11 ± 3.1 days, in laparoscopic or open approach, respectively. Sixteen (9.8%) and 57 (24.3%) postoperative complications occurred. CONCLUSION: Laparoscopy seems to be a safe and feasible approach for the treatment of acute incarcerated groin hernia. Further studies are required for definitive conclusions.

Bile detection of squamous cell carcinoma antigen (SCCA) in extrahepatic cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a rare biliary tract tumor with poor prognosis that often is challenging to diagnose and the majority of patients present with advanced stage. Squamous cell carcinoma antigen 1 (SCCA1) overexpression has been found in different tumors associated with poor prognosis and chemoresistance. AIMS: To assess the presence and possible prognostic role of SCCA1/2 isoforms in bile and serum of patients with CCA. METHODS: Forty seven surgical patients (36 with CCA and 11 with benign diseases) were prospectively included in the study. Serum and bile specimens were collected at the time of surgery and free and IgM-complexed SCCA was quantified by ELISA (Xeptagen, srl). RESULTS: Free or IgM linked SCCA was rarely found in serum, while SCCA was detectable in bile samples of patients with CCA, especially in those with extrahepatic form (43% vs 17%, p = 0.008), but not in controls. Despite similar tumor stage, these positive patients presented a trend toward a higher percentage of portal invasion (27% vs 15%) and of tumor recurrence than negative cases (62% vs 40%), although the difference was not statistically significant. CONCLUSION: These preliminary results indicate that bile testing for SCCA is a specific marker of extrahepatic CCA, with potential prognostic value.

Mineral bioaccessibility in 3D printed gels based on milk/starch/ĸ-carrageenan for dysphagic people.

Dysphagia is a condition that affects the ability to chew and swallow food and beverages, having a major impact on people's health and wellbeing. This work developed gel systems with a customized texture suitable for intake by dysphagic people using 3D printing and milk. Gels were developed using skim powdered milk, cassava starch (native and modified by the Dry Heating Treatment (DHT)), and different concentrations of kappa-carrageenan (ĸC). The gels were evaluated in relation to the starch modification process and concentration of gelling agents, 3D printing performance, and suitability for dysphagic people (following both the standard fork test described by the International Dysphagia Diet Standardization Initiative (IDDSI), and also using a new device coupled to a texture analyzer). Moreover, the best formulations were evaluated for mineral bioaccessibility through simulated gastrointestinal digestion based on INFOGEST 2.0 standardized method. The results showed that ĸC had a dominant effect compared to the DHT-modified starch on gel texture, 3D printing performance, and fork tests. The gels obtained by molding or 3D printing resulted in different behaviors during the fork test, which was associated with the gel extrusion process that breaks down their initial structure. The strategies applied to tailor the texture of the milk did not affect the mineral bioaccessibility, which was kept high (>80%).

Hypoglycemic effect of Coffea arabica leaf extracts and major bioactive constituents.

The present study focused on investigating the antioxidant, antiglycation activity, digestive enzymes inhibition, bioaccessibility and hypoglycemic effect of C. arabica leaves extracts. The extracts deactivated the O2•-, ROO•, H2O2, HOCl reactive oxygen species. Coffee leaves showed strong inhibition of α-glucosidase (IC50 = 40.30 µg mL-1) greater than the isolated metabolites and acarbose. There was also inhibition of pancreatic lipase (IC50 = 56.43 µg mL-1) in addition to a hypoglycemic effect in zebrafish similar to acarbose and metformin. With the exception of rutin, all biocompounds were detected at all stages of in vitro digestion. Finally, these results suggest that C. arabica leaf extracts possess antidiabetic and anti-obesity properties that can be attributed to the main metabolites and the synergistic action between them.Communicated by Ramaswamy H. Sarma.

Exposure to Mycobacterium avium decreases the protective effect of the DNA vaccine pVAXhsp65 against Mycobacterium tuberculosis-induced inflammation of the pulmonary parenchyma.

This work investigated the effect of previous Mycobacterium avium exposure on the protective ability of the DNA vaccine pVAXhsp65 against inflammation in the pulmonary parenchyma. BALB/c mice were presensitized with heat-killed M. avium and then immunized with three doses of pVAXhsp65 prior to challenge with Mycobacterium tuberculosis. M. avium sensitization induced high levels of spontaneous IL-5 production that were concomitant with a positive delayed-type hypersensitivity reaction; antigen-specific IFN-γ production was also observed upon splenocyte stimulation. Prior exposure to M. avium resulted in altered cytokine and antibody production induced by immunization with pVAXhsp65; instead of a Th1 response, vaccinated mice previously exposed to M. avium developed a strong Th2 response. This switch to a Th2 response coincided with the loss of the anti-inflammatory effect of pVAXhsp65 vaccination previously observed in the pulmonary parenchyma of mice infected with M. tuberculosis. These results suggest that exposure to environmental mycobacteria can modulate immune responses induced by mycobacterial vaccines other than bacillus Calmette-Guérin.

Experimental autoimmune encephalomyelitis evolution was not modified by multiple infections with Strongyloides venezuelensis.

According to the hygiene hypothesis, the increased incidence of allergic and autoimmune diseases in developed countries is mainly explained by the decreased contact between the human population and certain environmental agents as lactobacillus, mycobacteria and helminths. In this study, we evaluated the effect of multiple infections with Strongyloides venezuelensis on the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Multiple infections before EAE induction were not able to change the evolution of the disease. No alterations were observed in weight loss, clinical score and inflammation intensity at the central nervous system. The presence of significant levels of parasite-specific IgG1 but not IgG2b suggested a Th2 polarization. However, the percentage and absolute number of CD4+CD25+Foxp3+ T cells were not changed, being their levels in the spleen and lymph nodes of infected rats comparable to the ones found in normal animals. These results suggest that a Th2-polarized response without concomitant expansion of Foxp3+ regulatory T cells was not able to modify EAE progression. Even though these results do not threaten the hygiene hypothesis, they suggest that this paradigm might be an oversimplification. They also emphasize the need of a study to compare the immunoregulatory ability associated with different helminth spp.

Laparoscopic total mesorectal excision for rectal cancer: experience of a single center with a series of 174 patients.

BACKGROUND: Laparoscopic total mesorectal excision for low rectal cancer is not considered a gold standard treatment due to the high conversion rate and the long operation time. METHODS: A retrospective review examined a surgical series of 174 laparoscopic low rectal resections involving total mesorectal excision (1995-2006), with particular reference to technical points as well as surgical and oncologic outcomes. Miles operations and partial mesorectal excisions were excluded. RESULTS: The cancer affected the low rectum in 110 cases and the medium rectum in 64 cases. A total of 68 patients were subjected to neoadjuvant radiochemotherapy. The anastomosis was mechanical for 83.3% of the cases and intersphinteric through the perineum for 16.6% of the cases. Protective ileostomy was performed in 112 cases. The conversion rate was 4.6%. The mesorectum remained intact in 91.6% of the cases and was partially interrupted in 15 of the cases. In no case was it totally discontinued. The postoperative morbidity rate was 16.7%, and the mortality rate was 0.57%. The incidence of anastomotic fistulas was 14.4%. The percentage was higher for males (18.6 vs 8.3%) and correlated with the low distance of the tumor from the anal verge (18.2 vs 7.8%) and the absence of a protection ileostomy (20.9 vs 10.7%). After an average follow-up period of 48.6 months (range, 24-149 months), six patients (3.44%) experienced a pelvic recurrence. The 5-year overall survival rate was 75.4%, and the disease-free survival rate was 61.9%. CONCLUSION: Laparoscopic total mesorectal excision for low rectal cancer is safe and effective, allowing surgical and oncologic outcomes similar to those reported for open surgery.

Superhydrophilic and tribological improvements of polymeric surfaces via plasma enhanced chemical vapor deposition ceramic coatings.

The main object of this study is the treatment of polymeric (PVC, PC) surfaces with the aim of inducing enhanced superhydrophilic characteristics together with nanohardness features; this would allow polymeric surfaces to have longer durability and prevent the accumulation of dirt on the surface which could disable the proper use of these polymeric surfaces. Indeed plastic surfaces are difficult substrates to be covered effectively and functionalized, mainly due to their high sensitivity to heat treatments and irradiation in the UV-Vis range together with their inert behavior. Their functionalization is achieved through the deposition of ceramic coatings such as titania (TiO2), on the polymeric surfaces via PECVD (Plasma Enhanced Chemical Vapor Deposition) at low temperatures. Characterizations are carried out by contact angle analysis for the superhydrophilic characteristics, and by nanoindentation analysis for the tribological features. A cold PECVD discontinuous method allowed us to improve nanohardness, reaching a value of 1.39 GPa which is nearly ten times higher than that of the uncoated polymeric substrate, and seems a promising solution for improving uniformity of the coatings. Superhydrophilic behavior of the activated TiO2 surfaces showed contact angle values lower than 10 degrees.

Validation of a documentary on Chagas disease by a population living in an endemic area.

Educational interventions may trigger actions that contribute to prevent parasitic diseases, such as Chagas disease (CD). This study aimed at investigating the impact of an instructional video named "Documentary on Chagas Disease" on knowledge about CD and its vectors displayed by a population that lives in an endemic area in Brazil, so as to validate it as an educational tool. The video was shown to 226 subjects, divided into two groups. Group 1 was composed of users of Basic Health Units (BHU) in Pelotas and Pinheiro Machado, cities located in Rio Grande do Sul (RS) state, Brazil, where CD is endemic. Group 2 consisted of students who attend three public schools located in the rural area in Pinheiro Machado, RS. Two questionnaires with questions about their knowledge about triatomines and CD were applied, before and after the documentary was shown. After the video was shown, there was significant increase in individuals' knowledge (in both groups) about "kissing bugs", their notification, the disease and its prevention. Besides, watchers considered that the quality of the material was satisfactory. Since the "Documentary on CD" can be easily accessed on the internet and was effective in teaching the population that lives in endemic areas, its use should be encouraged in places and meetings connected to health that aim at fighting against triatominae and at exposing an updated view of CD.

Should enhanced recovery after surgery (ERAS) pathways be preferred over standard practice for patients undergoing abdominal wall reconstruction? A systematic review and meta-analysis.

PURPOSE: Although many studies assessing enhanced recovery after surgery (ERAS) pathways in abdominal wall reconstruction (AWR) have recently demonstrated lower rates of postoperative morbidity and a decrease in postoperative length of stay compared to standard practice, the utility of ERAS in AWR remains largely unknown. METHODS: A systematic literature search for randomized and non-randomized studies comparing ERAS (ERAS +) pathways and standard protocols (Control) as an adopted practice for patients undergoing AWR was performed using MEDLINE, the Cochrane Central Register of Controlled Trials, Scopus, Web of Science, and EMBASE databases. A predefined search strategy was implemented. The included studies were reviewed for primary outcomes: overall postoperative morbidity, abdominal wall morbidity, surgical site infection (SSI), and length of hospital stay; and for secondary outcome: operative time, estimated blood loss, time to discontinuation of narcotics, time to urinary catheter removal, time to return to bowel function, time to return to regular diet, and readmission rate. Standardized mean difference (SMD) was calculated for continuous variables and Odds Ratio for dichotomous variables. RESULTS: Five non-randomized studies were included for qualitative and quantitative synthesis. 840 patients were allocated to either ERAS + (382) or Control (458). ERAS + and Control groups showed equivalent results with regard to the incidence of postoperative morbidity (OR 0.73, 95% CI 0.32-1.63; I2= 76%), SSI (OR 1.17, 95% CI 0.43-3.22; I2= 54%), time to return to bowel function (SMD - 2.57, 95% CI - 5.32 to 0.17; I2= 99%), time to discontinuation of narcotics (SMD - 0.61, 95% CI - 1.81 to 0.59; I2= 97%), time to urinary catheter removal (SMD - 2.77, 95% CI - 6.05 to 0.51; I2= 99%), time to return to regular diet (SMD - 0.77, 95% CI - 2.29 to 0.74; I2= 98%), and readmission rate (OR 0.82, 95% CI 0.52-1.27; I2= 49%). Length of hospital stay was significantly shorter in the ERAS + compared to the Control group (SMD - 0.93, 95% CI - 1.84 to - 0.02; I2= 97%). CONCLUSIONS: The introduction of an ERAS pathway into the clinical practice for patients undergoing AWR may cause a decreased length of hospitalization. These results should be interpreted with caution, due to the low level of evidence and the high heterogeneity.

Neonatal BCG immunization followed by DNAhsp65 boosters: highly immunogenic but not protective against tuberculosis - a paradoxical effect of the vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Rapid quantification of the effects of blotting for correlation of light and cryo-light microscopy images.

Recent technical developments allowed the accurate correlation of fluorescently labelled organelles in living cells to cryo-electron micrographs. We aimed at expanding this approach to Plasmodium berghei sporozoites, the motile forms of a rodent malaria parasite, which can be imaged by cryo-electron tomography in toto without the need for sectioning. Sporozoites are crescent shaped eukaryotic cells that move on flat supports including EM grids in a circular, unidirectional manner. While sporozoites can be visualized with fluorescent light and cryo-light microscopy prior to tomography, few motile sporozoites remained on the grid after blotting excess liquid impairing a complete correlation from light microscopy to cryo-electron tomography. Comparison with cells showing different adhesion strengths demonstrated that the ratio of cells remaining on the grid can be rapidly determined, but that the integrity of the cells has to be carefully monitored as the blotting applies high physical stress to the cells. We demonstrate a quick technique to assess not only feasibility of direct correlation without fixation but also the damage caused by blotting.