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With the previous worldwide initial coronavirus disease 2019 (COVID-19) pandemic, a notable rise in spontaneous pneumomediastinum with/without pneumothorax (SPP) has been noted. Most cases were initially reported as complications secondary to barotrauma from mechanical ventilation (MV) with COVID-19. However, with the Delta strain, starting from December 2020, there have been multiple reports of SPP. The SPP is an uncommon complication outside use of assisted ventilation with either noninvasive positive pressure ventilation (NIPPV) or MV. COVID-19 has been linked to higher incidence of SPP without use of NIPPV or MV. We present a series of 5 cases with a polymerase chain reaction–confirmed COVID-19 diagnostic testing whose hospital course was complicated by SPP unrelated to the use of either NIPPV or MV.

Research Question: What was the utilization, effectiveness and safety of assisted reproductive technology (ART) in Latin America during 2020?, Design: Retrospective collection of multinational data on ART performed by 188 institutions in 16 countries., Results: Overall, 87,732 initiated cycles resulted in 12,778 deliveries and 14,582 births. The major contributors were Brazil (46.0%), Mexico (17.0%) and Argentina (16.8%). However, the highest utilization (cycles/million inhabitants) was Uruguay with 558, followed by Argentina with 490 and Panama with 425 cycles/million. Globally, women aged ≥40 years increased to 34% while women ≤34 dropped to 24.7%. After removing freeze-all cycles, the delivery rate per oocyte retrieval was 14.8% for intracytoplasmic sperm injection and 15.6% for IVF. Single-embryo transfer (SET) represented 38.3% of all fresh transfers, with delivery rate per transfer of 20.0%; this increased to 32.4% for elective SET (eSET) and 34.2% for blastocyst eSET, compared with blastocyst elective double embryo transfer (eDET) of 37.9%. However, multiple births increased from 1% in eSET to 30.5% in eDET. Perinatal mortality increased from 7.7‰ in singletons to 24.4‰ in twins and 64.0‰ in triplets. Frozen embryo transfer (FET) represented 66.6% of all transfers, with a delivery rate/transfer of 29.0%, significantly higher than 23.9% after fresh transfers at all ages (p<0.0001). Preimplantation genetic testing, reported in 8920 cycles, significantly improved delivery rate and decreased miscarriage rates at all ages (p≤0.041), including oocyte donation (p=0.002). Endometriosis was diagnosed in 28.3% of cases. The delivery rate in 5779 women after removal of peritoneal endometriosis was significantly better than tubal and endocrine factors in women aged 35-39 (p=0.0004) and women aged ≥40 (p=0.0353)., Conclusions: Systematic collection and analysis of big data in a south-south cooperation model allow regional growth by implementing evidence-based reproductive decisions.

Research Question: What was the utilization, effectiveness and safety of assisted reproductive technology (ART) in Latin America during 2019?, Design: This was a retrospective collection of multinational data on ART performed at 196 institutions from 15 countries., Results: A total of 106,918 initiated cycles, 18,133 deliveries and 21,096 births were reported. ART utilization was 24-558 cycles per million inhabitants. Women aged ≥40 years represented 32.9% of fresh IVF and intracytoplasmic sperm injection (ICSI) cycles. After removing freeze-all cycles, the delivery rate per oocyte retrieval was 17.3% for ICSI and 19.5% for IVF. Overall, single-embryo transfer (SET) represented 36.2% of fresh transfers, with a 19.5% delivery rate per transfer, increasing to 30.7% for elective SET and 32.7% for blastocyst elective SET (eSET). The delivery rate for double-embryo transfers (DET) was 27.8%, increasing to 37.1% after elective DET. This 6.4% increment in deliveries between eSET and elective DET resulted in a 12-fold increase in twin births. Furthermore, overall perinatal mortality was more than two-fold higher for twin compared with singleton deliveries. The delivery rate for frozen-thawed SET reached 28.1%, most being blastocyst transfers. Of all births, 72.3% were singletons, 26.4% twins and 1.3% triplets and higher multiples. Preterm deliveries reached 14.3% for singletons and 58.1% for twins. Perinatal mortality was 7.4‰ in singletons, 17.2‰ for twins and 62.9‰ for triplets or higher., Conclusions: The number of initiated cycles has slowly increased in countries with laws or regulations facilitating access. FET cycles predominate and blastocyst SET are also increasing. The data show that, especially in young women and oocyte recipients, when there is more than one blastocyst for transfer, eSET should be the rule.

Objective: What are the trends in patient characteristics, effectiveness and safety of assisted reproductive technology (ART) performed in Latin America over the past three decades, as well as the detailed outcomes of procedures initiated in 2018?., Design: Retrospective collection of multinational data including epidemiology and outcomes of ART performed between 1990 and 2018., Results: Over these 30 years we report 955,117 initiated cycles, 191,191 deliveries and 238,045 live births. In 1990, 66.5% of women were ≤34 years and 8.7% ≥40 years; in 2018, 26.4% of women were ≤34 years and 32.0% were ≥40 years. In 1990, 60.4% of transfers included ≥3 embryos, falling to 13.5% in 2018, and single embryo transfer (SET) increased from 13.8% to 30.4% between 1990 and 2018. Delivery rate per fresh transfer increased from approximately 17% in the 1990s to 25% in 2018, with a meaningful drop in high-order multiples, from 5-9% in the 1990s to 0.4% in 2018. This drop is associated with increasing use of frozen embryo transfer (FET) (57% in 2018) compared with 10% in 2000. In 2018, delivery rate in FET was 28.3%, reaching 31.2% in freeze-all cycles; and the cumulative live birth rate (fresh + FET) was 41.9%. Elective SET also increased, from 0.9% in 2010 to 10% in 2018. The delivery rate in elective SET (31.7%) was only 5.4% lower than elective double embryo transfer (DET) (37.1%); however, multiple births increased from 2.1% to 25.5% twins and 0.4% triplets in elective DET., Conclusions: The Latin American Registry of Assisted Reproduction (RLA) celebrates 30 years of voluntary reporting from a total of nearly 200 centres in 15 countries. This South-South Cooperation network has proven to be an efficient and safe system for technological transfer and regional growth.

Research Question: What was the utilization, effectiveness and safety of assisted reproductive techniques performed in Latin America during 2017., Design: Retrospective collection of multinational data on ART performed in 188 institutions from 15 Latin American countries., Results: We are reporting 93,600 initiated cycles, 16,976 deliveries and the birth of 20,404 babies. ART utilization was 221 cycles/million inhabitants (15 to 535). Despite women aged ≥40 represented 30.5% of fresh IVF/ICSI, after removing freeze-all cycles, delivery rate per oocyte retrieval was 19.9% for ICSI and 20.2% for IVF. Overall, single embryo transfer (SET) represented 26.9% of fresh transfers, with 18.2% delivery rate per transfer; increasing to 32.3% in elective SET. Delivery rate in double embryo transfers (DET) was 28.3% increasing to 37.3% with elective DET. This 5% increment in births in eDET over eSET resulted in10-fold increase in twin births, almost 3 weeks' shorter gestations and 3-fold increase in perinatal mortality. Delivery rate in frozen/thawed SET, reached 25.5% increasing to 30.8% with DET; the majority being blastocysts transfers. Of all births, 67% were singletons, 31.4% twins, and 1.6% triplets and higher. Overall, preterm deliveries reached 9.5% in singletons, 64.3% in twins and 97.9% in triplets; and perinatal mortality was 9.4‰ in singletons, 25.3‰ in twins, and 63.3‰ in high-order multiples., Conclusions: The number of initiated cycles slowly increases. Frozen embryo transfers, blastocyst transfers and SET are also increasing. Our data shows that especially in young women and oocyte recipients, when there is more than one blastocyst for transfer, elective SET should be the rule.

Research Question: What was the utilization, effectiveness and perinatal outcome of assisted reproductive technologies (ART) performed in Latin America during 2016?, Design: Retrospective collection of multinational data on ART performed in 178 institutions from 15 Latin American countries., Results: This paper reports on 85,474 initiated cycles, 15,070 deliveries and 18,182 babies born in this period. Of all fresh autologous IVF/intracytoplasmic sperm injection (ICSI) cycles, 40.9% were performed in women aged 35-39 years, and 31.1% in women aged ≥40 years. After removing freeze-all cycles, delivery rate per oocyte retrieval was 20.31% for ICSI and 21.85% for IVF. Fresh single embryo transfer including all age categories represented 22.96%, with a 15.35% delivery rate per transfer. Double embryo transfer represented 61.58% of transfers, with a 27.62% delivery rate per transfer. Multiple births included 18.12% twins and 0.55% triplets and higher. In oocyte donation, delivery rate per transfer was 32.89%, with a twin and triplet rate of 23.48% and 0.73%, respectively. Overall, preterm deliveries reached 17.11% in singletons, 65.69% in twins and 95.51% in triplets. Perinatal mortality was 8.0‰ in singletons, 19.0‰ in twins, and 62.3‰ in high-order multiples., Conclusions: The number of initiated cycles continues to increase. Compared with previous years, the number of embryos transferred decreased while the proportion of single embryo transfers increased with a drop in multiple births. It is vital to motivate health care providers and consumers to continue this trend., (Copyright © 2019. Published by Elsevier Ltd.)

Objective: Preeclampsia (PE) occurs in 4.6% of pregnancies worldwide. The social phenomenon of increasing maternal age has raised the demand for donor oocytes. Egg donation has allowed women with poor ovarian reserve, premature ovarian failure, genetic disorders or surgical menopause to get pregnant. Recipients provide a unique model of immune response because of the differences in the genetic makeup of mothers and fetuses. In PE, immune tolerance may be impaired as a result of having non-autologous eggs implanted. Egg donation is a highly successful assisted reproductive technology, despite the significant number of issues arising from the implantation of non-autologous eggs. This study aimed to determine whether there is an association between egg donation and preeclampsia., Methods: A systematic review of the literature available in PubMed and Google Scholar was carried out from January of 1995 to August of 2016 using the terms 'oocyte donation, preeclampsia', 'oocyte donation, in vitro fertilization, preeclampsia', 'oocyte donation, preeclampsia, outcomes pregnancies', 'oocyte donation, obstetric outcome.' Only six retrospective cohort studies met the selection criteria., Results: The meta-analysis revealed a statistically significant association between egg donation and onset of preeclampsia (OR 4.50; 95% CI: 3.28-6.19; p<0.0001)., Conclusion: Oocyte donation is associated with increased risk of preeclampsia in singleton pregnancies. Therefore, it is crucial to properly record and assess this finding when egg donation is the chosen assisted reproductive technology to attain pregnancy.

Multinational data on assisted reproduction techniques undertaken in 2014 were collected from 159 institutions in 15 countries in Latin America. Treatments included IVF/ ICSI, FET, OD, PGD and fertility preservation (FP). 41.34% of IVF/ICSI cycles were performed in women aged 35 to 39 years and 23.35% in women aged 40 and older. After removing cases with total freezing, delivery rate per oocyte retrieval was 25.05% for ICSI and 27.41% for IVF. Multiple births included 20.78% twins and 0.92 % triplets and over. In OD, twins reached 28.93% and triplets 1.07 %. Preterm deliveries reached 16.4% in singletons, 55.02% in twins and 76% in triplets. Perinatal mortality in 18,162 births was 23 per 1000 in singletons, 35 per 1000 in twins, and 36 per 1000 in high-order multiples. Elective single embryo transfer (eSET) represented only 2.63 % of fresh transfers, with a delivery rate of 32.15% per transfer. Elective double embryo transfer (eDET) represented 23.74% of transfers, with a delivery rate of 41.03% per transfer. Among babies born during this period 11,373 (62.6%) were singletons; 6,398 (35.2%) twins, and 391 (2.2%), triplets and more. Given the effect of multiple births on prematurity, morbidity and perinatal mortality, reinforcing the existing trend of reducing the number of embryos transferred is mandatory.

Objective: The main objective of this study was to assess the prevalence of overweight and obesity among patients undergoing assisted reproductive technology (ART) in Latin America and its consequences on treatment outcomes., Methods: We used the Latin American Registry of ART to obtain women's age and body mass index (BMI), cancellation rate, number of oocytes retrieved and embryos transferred, clinical pregnancy, live birth and miscarriage rates from 107.313 patients undergoing autologous IVF and ICSI during four years; a multivariable analysis was performed to determine the effect of BMI on cancellation, oocytes retrieved, pregnancy, live birth and miscarriage, adjusting for age, number of embryos transferred and embryo developmental stage upon embryo transfer, when appropriate., Results: The prevalence of overweight and obesity was 16.1% and 42.4%, respectively; correcting for age of female partner, overweight and obesity were associated to an increase in the odds of cancellation and to a lower mean number of oocytes retrieved; after adjusting for age, number of embryos transferred and stage of embryo development at transfer, we found that the BMI category was not associated to a change in the likelihoods of pregnancy, live birth and miscarriage., Conclusions: The prevalence of obesity among women seeking ART in Latin America is surprisingly high; however, BMI does not influence the outcome of ART performed in these women.

Objective: To determine whether the addition of either urinary or recombinant LH in patients undergoing routine clinical care improved the outcome in terms of the number of oocytes recovered for insemination or the delivery rate per initiated cycle., Design: Cohort analysis., Patient(s): Couples undergoing IVF/ICSI in 158 institutions in 15 countries in Latin America., Setting: In vitro fertilization clinics., Intervention(s): We compared the outcome of three different protocols of COH, including rFSH only, rFSH plus rLH, and rFSH plus hMG., Main Outcome Measure(s): The number of mature oocytes recovered and inseminated; proportion of ETs at the blastocyst stage; clinical pregnancy, miscarriage, and delivery rates; proportion of cycles with embryo cryopreservation; and mean number of embryos cryopreserved., Result(s): After correcting for the age of the female partner, body mass index, number of embryos transferred, and stage of embryo development at transfer, we found that LH addition was not associated with an increase in the mean number of metaphase II oocytes inseminated or with an increase in the delivery rate or changes in the miscarriage rate., Conclusion(s): Our study strongly suggests that in routine clinical practice, the type of controlled ovarian stimulation-FSH alone or in combination with LH-has little impact on the outcome of assisted reproductive technology; therefore a more friendly and accessible alternative should be favored., (Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Multinational data on assisted reproduction techniques undertaken in 2013 were collected from 158 institutions in 15 Latin American countries. Individualized cycle-based data included 57,456 initiated cycles. Treatments included autologous IVF and intracytoplasmic sperm injection (ICSI), frozen embryo transfers, oocyte donations. In autologous reproduction, 29.22% of women were younger than 35 years, 40.1% were 35-39 years and 30.6% were 40 years or older. Overall delivery rate per oocyte retrieval was 20.6% for ICSI and 25.4% for IVF. Multiple births included 20.7% for twins and 1.1% for triplets and over. In oocyte donations, twins reached 30% and triplets 1.4%. In singletons, pre-term births were 7.5%: 36.58% in twins and 65.52% in triplets. The relative risk for prematurity was 4.9 (95% CI 4.5 to 5.3) in twins and 8.7 (95% CI 7.6 to 10.0) in triplets and above. Perinatal mortality was 29.4 per 1000 in singletons, 39.9 per 1000 in twins and 71.6 per 1000 in high order multiples. Elective single embryo transfer represented only 2% of cycles, with delivery rate of 39.1% in women aged 34 years or less. Given the effect of multiple births and prematurity, it is mandatory to reduce the number of embryos transferred in the region., (Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Embryo cryopreservation is an integral part of assisted reproduction techniques; it allows the sequential transfer of all embryos, thus diminishing the risk of multiple pregnancies and associated perinatal complications. To address concerns about the safety of this procedure, neonatal outcome after 43,070 fresh embryo transfers was compared with 12,068 frozen-thawed embryo transfers (FET). After adjusting for maternal age, gestational age, embryo development at time of transfer, number of babies born and gestational order, FET was not found to be associated with an increase in perinatal mortality (odds ratio [OR] 1.72, 95% confidence interval [CI] 0.81 to 3.62); preterm birth (OR 1.05, 95% CI 0.93 to 1.18); or extreme preterm birth (OR 0.82, 95% CI 0.64 to 1.06). Furthermore, after correcting for known confounding factors, FET was found to be associated with an increase in neonatal weight of 39.7 g (95% CI 1.54 to 64.10; P < 0.0001). Embryo cryopreservation was, therefore, not associated with an increase in the risk of poor perinatal outcome., (Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Multinational data on assisted reproduction technologies were collected from 155 institutions in 14 Latin American countries during 2012. Case-by-case data included 47,326 assisted reproduction technology cycles covering over 80% of cycles carried out in Latin America. Treatments included IVF, intracytoplasmic sperm injection (ICSI), frozen embryo transfers, oocyte donations and fertility preservation. Embryo transfer and IVF-ICSI was carried out in 39% of women aged 35-39 years and 31% of women aged 40 years or over. Delivery rate per oocyte retrieval was 20.9% for ICSI and 26.5% for IVF. Multiple births comprised 20.6% twins and 1.2% triplets and over. In oocyte donations, twins reached 27.8% and triplets and over 2.4%. Pre-term births in singletons were 14%. The relative risk of prematurity increased by 4.30 (95% CI 4.1 to 4.6) in twins and 43.8 (95% CI 28.5 to 67.4) in triplets and higher. Perinatal mortality increased from 25.2 per thousand in singletons to 44.4 in twins and 80.7 in triplets and over. Elective single embryo transfer was carried out in only 1.4%, of cycles, with a delivery rate of 30% in women 34 years or younger, and should be considered the way forward provided access is facilitated with public funding., (Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Objective: This report examines information on Assisted Reproduction Technologies performed in Latin America (LA) during 2012., Methods: Multinational data were collected directly from 155 institutions in 14 countries. Individualized, case-bycase data include 47,326 ART cycles covering more than 80% of cycles performed in LA. Treatments included in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), frozen embryo transfers (FET), oocyte donations (OD) and fertility preservation., Results: In 39% of ET IVF/ICSI was performed in women age 35-39 and 31% in women ≥40 years. Delivery rate (DR) per pick-up (OPU) in ICSI and IVF cycles, were 20.9% and 26.5%, respectively. Overall multiple births comprised 20.6% twins and 1.2% triplets. Furthermore, in OD, twins and triplets reached 27.8% and 2.4%, respectively. Pre term births in singletons were 14%. The relative risk of prematurity increased by 4.30 (95% CI 4.1-4.6) in twins, and 43.8 (95% CI 28.5-67.4) in ≥ triplets. Perinatal mortality increased from 25.2‰ in singletons, to 44.0‰ in twins and 80‰ in ≥ triplets. Elective single embryo transfer (eSET) was performed in only 1.4% of cycles with DR of 30% in women ≤34 years., Conclusion: Trends over the last 20 years show that eSET should be the way to go provided access is facilitated with public funding.

As part of Assisted Reproductive Technologies (ART), the advent of embryo freezing lowered the number of embryos transferred, decreasing multiple births without jeopardizing pregnancy rates. Using vitrification technology, 90% of embryos survive after thawing, producing clinical pregnancy rates similar to those of fresh embryos (41.6%y 44.3% respectively). Furthermore, cumulative pregnancy rates, obtained after transferring fresh plus frozen/thawed embryos, can reach 70%. Frozen embryo transfers (FET) are reported by six of seven institutions, which are part of the Chilean ART registry, and altogether constitute 22.8% of all ART procedures. Increasing use of cryopreservation lowered overall multiple gestations from 33% in 1995 to 23% in 2011, reducing pre term births and perinatal mortality. For many people, embryo freezing generates ethical dilemmas, due to the potential risks to which embryos are exposed, and the uncontrolled accumulation and disposal of human embryos. Scientific evidence today shows that frozen/thawed embryos are not exposed to disproportionate risks, and by hindering its use, both women and their children are exposed to the risks of multiple gestation, repeated cycles of ovarian hormonal stimulation or the impossibility to afford repeated ART cycles. In this article, we provide biomedical, as well as ethical, arguments to sustain that embryo cryopreservation is not only justified but fundamental when offering infertility treatment with ART.

Objective: To identify a gene expression signature in human cumulus cells (CCs) predictive of pregnancy outcome across multiple clinics, taking into account the clinic and patient variations inherent in IVF practice., Design: Retrospective analysis of single human cumulus-oocyte complexes with the use of a combined microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) approach., Setting: Multiple private IVF clinics., Patient(s): Fifty-eight patients. Samples from 55 patients underwent qRT-PCR analysis, and samples from 27 patients resulted in live birth., Intervention(s): Gene expression analysis for correlation with pregnancy outcome on individual human CCs collected immediately after oocyte retrieval. Pregnancy prediction analysis used leave-one-out cross-validation with weighted voting., Main Outcome Measure(s): Combinatorial expression of 12 genes in 101 samples from 58 patients., Result(s): We found a set of 12 genes predictive of pregnancy outcome based on their expression levels in CCs. This pregnancy prediction model had an accuracy of 78%, a sensitivity of 72%, a specificity of 84%, a positive predictive value of 81%, and a negative predictive value of 76%. Receiver operating characteristic analysis found an area under the curve of 0.763 ± 0.079, significantly greater than 0.5 (random chance prediction)., Conclusion(s): Gene expression analysis in human CCs should be considered in identifying oocytes with a high potential to lead to pregnancy in IVF-ET., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Screens for compounds and proteins with anti-cancer activity employ viability assays using relevant cancer cell lines. For leukaemia studies, the human leukaemia cell line, HL-60, is often used as a model system. To facilitate the discovery and investigation of anti-leukaemia therapeutics under physiological conditions, we have engineered HL-60 cells that stably express firefly luciferase and produce light that can be detected using an in vivo imaging system (IVIS). Bioluminescent HL-60luc cells could be rapidly detected in whole blood with a sensitivity of approximately 1000 viable cells/200 microl blood. Treatment of HL-60luc cells with the drug chlorambucil revealed that the bioluminescent viability assay is able to detect cell death earlier than the Trypan blue dye exclusion assay. HL-60luc cells administered intraperitoneally (i.p.) or intravenously (i.v.) were visualized in living mice. The rapidity and ease of detecting HL-60luc cells in biological fluid indicates that this cell line could be used in high-throughput screens for the identification of drugs with anti-leukaemia activity under physiological conditions., ((c) 2007 John Wiley & Sons, Ltd.)

Aggregatibacter actinomycetemcomitans is an oral bacterium that causes localized aggressive periodontitis (LAP) and extra-oral infections such as sub-acute infective endocarditis. As part of its array of virulence factors, A. actinomycetemcomitans produces leukotoxin (LtxA), a member of the RTX family of toxins. LtxA kills human leukocytes and we have recently shown that the toxin is required for beta-hemolysis by A. actinomycetemcomitans on solid medium. In other RTX toxin-producing bacteria, an outer membrane channel-forming protein, TolC, is required for toxin secretion and drug export. We have identified an ORF in A. actinomycetemcomitans that encodes a putative protein having predicted structural properties similar to TolC. Inactivation of this ORF resulted in a mutant that was no longer beta-hemolytic and did not secrete LtxA. This mutant was significantly more sensitive to antimicrobial agents compared to the wild type strain and was unable to export the antimicrobial agent berberine. Thus, this ORF was named tdeA for "toxin and drug export". Examination of the DNA sequence surrounding tdeA revealed two upstream ORFs that encode proteins similar to the drug efflux proteins, MacA and MacB. Inactivation of macB in A. actinomycetemcomitans did not alter the drug sensitivity profile or the hemolytic activity of the mutant. The genes macA, macB and tdeA are organized as an operon and are constitutively expressed as a single transcript. These results show that A. actinomycetemcomitans indeed requires a TolC-like protein for LtxA secretion and that this protein, TdeA, also functions as part of a drug efflux system.

The gram-negative oral and systemic pathogen Aggregatibacter (Actinobacillus) actinomycetemcomitans produces a leukotoxin (LtxA) that is a member of the RTX (repeats in toxin) family of secreted bacterial toxins. We have recently shown that LtxA has the ability to lyse erythrocytes, which results in a beta-hemolytic phenotype on Columbia blood agar. To determine if LtxA is regulated by iron, we examined beta-hemolysis under iron-rich and iron-limiting conditions. Beta-hemolysis was suppressed in the presence of FeCl3. In contrast, strong beta-hemolysis occurred in the presence of the iron chelator deferoxamine. We found that secretion of LtxA was completely inhibited by free iron, but expression of ltxA was not regulated by iron. Free chromium, cobalt, and magnesium did not affect LtxA secretion. Other LtxA-associated genes were not regulated by iron. Thus, iron appears to play an important role in the regulation of LtxA secretion in A. actinomycetemcomitans in a manner independent of gene regulation.

Actinobacillus actinomycetemcomitans is the etiologic agent of localized aggressive periodontitis, a rapidly progressing oral disease that occurs in adolescents. A. actinomycetemcomitans can also cause systemic disease, including infective endocarditis. In early work on A. actinomycetemcomitans workers concluded that this bacterium is not beta-hemolytic. More recent reports have suggested that A. actinomycetemcomitans does have the potential to be beta-hemolytic. While growing A. actinomycetemcomitans on several types of growth media, we noticed a beta-hemolytic reaction on media from one manufacturer. Beta-hemolysis occurred on Columbia agar from Accumedia with either sheep or horse blood, but not on similar media from other manufacturers. A surprising result was that mutants of A. actinomycetemcomitans defective for production of leukotoxin, a toxin that is reportedly highly specific for only human and primate white blood cells, are not beta-hemolytic. Purified leukotoxin was able to lyse sheep and human erythrocytes in vitro. This work showed that in contrast to the accepted view, A. actinomycetemcomitans leukotoxin can indeed destroy erythrocytes and that the production of this toxin results in beta-hemolytic colonies on solid medium. In light of these results, the diagnostic criteria for clinical identification of A. actinomycetemcomitans and potentially related bacteria should be reevaluated. Furthermore, in studies on A. actinomycetemcomitans leukotoxin workers should now consider this toxin's ability to destroy red blood cells.

The ATP-dependent Lon protease is a multi-functional enzyme that is conserved from archae to mammalian mitochondria, which not only degrades protein substrates but also binds DNA. As a starting point toward understanding Lon function in development, the mouse Lon cDNA was cloned and the encoded protein was characterized in cultured mammalian cells, in yeast and in vitro. Mouse Lon shows 87, 40 and 33% amino acid similarity with the human, yeast and bacterial homologs, respectively. Expression of a single mouse Lon transcript is detected in liver>heart>kidney>testis and is present during early embryonic development. Endogenous as well as transiently overexpressed mouse Lon co-localize with mitochondrial markers and have half-lives greater than 24 h as determined by pulse-chase studies. Enzymatically active mouse Lon that hydrolyses ATP and degrades protein and peptide substrates in an ATP-dependent manner also specifically binds to single-stranded but not to double-stranded DNA oligonucleotides. We propose that binding to TG-rich DNA sequences has been conserved between the mouse and human proteins. In addition, the evolutionary conservation of mitochondrial Lon function is demonstrated by the ability of mouse Lon to substitute for the yeast protein in vivo.

Background: We attempted to determine factors contributing to the extent of initial curative resection for colon cancer in a population-based cohort. Total abdominal colectomy with ileorectal anastomosis (TAC-IR) may be considered for young patients or those with a colorectal cancer family history to prevent metachronous lesions and facilitate surveillance., Methods: All Ontario patients newly diagnosed with colon cancer over 12 months beginning in July 1997 were staged at the time of surgery. The extent of resection was compared with variables, including familial risk obtained from the Ontario Familial Colon Cancer Registry., Results: Complete staging was possible for 86% of patients. A total of 1223 patients had a potentially curative resection: 17%, 46%, and 36% were stage I, II, and III, respectively. Patients were more likely to receive a TAC-IR if they were < or = 50 years old (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.8-6.6), if they had a synchronous lesion (OR, 28.37; 95% CI, 12.2-61.2), or if they were at a teaching hospital (OR, 2.8; 95% CI, 1.6-4.7), but not if they had a family history (OR,.7; 95% CI,.3- 1.5)., Conclusions: Young age, teaching hospital, and multiple cancers but not family history were important factors for performing a TAC-IR.

Background: Quality end-of-life care is an increasing concern for the public and the medical profession. Surgical textbooks could serve as an important educational and reference resource to improve this care., Methods: Four general surgical textbooks were scored for helpful information on death and dying for eight surgical diseases. For each disease, nine content domains related to care of the dying patient were evaluated. Three texts included a chapter on cancer that was evaluated separately., Results: Disease epidemiology, prognosis/prevention, progression, and medical interventions were generally well discussed in all textbooks. However, little helpful information was provided with regards to breaking bad news/advanced care planning, mode of death, treatment decision-making, effect on family/surgeon, and symptom management. Cancer chapters also addressed only a few of these concerns., Conclusion: Death and the dying patient are sufficiently frequent in surgical practice that it would be appropriate to increase the amount of information provided.

Background: Malignant gastrointestinal stromal tumors (M-GIST) are rare mesenchymal tumors originating in the wall of the gastrointestinal (GI) tract. Previous studies have included limited numbers of patients, and most included malignant and benign cases from throughout the GI tract. We reviewed the experience of a single tertiary cancer care center with M-GIST of the small intestine only., Methods: A prospective database identified all patients seen from 1989 to 1998. Clinical and pathological data, treatment, and outcome were analyzed. Overall median follow-up time was 24 months (range, 1-176 months)., Results: Fifty patients (31 male, 19 female) were identified. Mean age at diagnosis was 55 years. Disease was localized in 11 patients, locally advanced (invasion into adjacent organs/peritoneum) in 24 patients, perforated in 4 patients, multiple primary lesions in 2 patients, and distant metastases in 9 patients. All patients underwent resection, which was complete in 70%. Locoregional recurrence (LR) developed in 43% (median, 25 months), and distant metastases in 59% (median, 21 months) of patients at risk. At last follow-up, 14 patients were alive (6 disease-free), 2 had died disease-free, and 34 died with recurrent disease. Overall survival (OS) was similar for localized and locally advanced disease; OS also was similar for patients with multiple primaries and distant metastases at diagnosis. Patients were grouped into three stages: (I) patients with localized and locally advanced disease; (II) patients with perforated; and (III) patients with multiple primaries and distant metastases. Actuarial OS at 5 years was 41% (n = 50)--42% for those with complete resection and 8% for incomplete resection. Univariable analysis showed that earlier stage at diagnosis (P = .001) and completeness of resection (P = .004) predicted for longer OS., Conclusions: Most patients with M-GIST of the small intestine relapse following resection, but survival may be prolonged. In univariable analysis, stage at presentation and complete resection were significant prognostic variables for OS; grade was not significant. Localized and locally advanced M-GIST of the small intestine have a mean OS > 5 years. Complete resection should be the goal of initial surgical treatment.

The transcriptional activator Ste12p is required for the expression of genes induced by mating pheromone in the yeast Saccharomyces cerevisiae. We identified mutations in the amino-terminal DNA-binding domain of Ste12p that lead to constitutively high-level transcription of pheromone-induced genes. The behaviour of these mutant proteins is consistent with an enhanced DNA-binding ability. Cells carrying these hyperactive proteins retain their sensitivity to pheromone treatment, and their phenotype is largely dependent on the presence of at least one of the MAP kinases (Fus3p or Kss1p) and the scaffold protein Ste5p. Deletion of either FUS3 or KSS1 leads to a marked increase in Ste12p activity, consistent with a negative regulatory role for Fus3p, similar to that described for Kss1p. The properties of the constitutive mutants support the idea that the pheromone response pathway plays a role in basal as well as pheromone-induced transcription., (Copyright 2000 John Wiley & Sons, Ltd.)

So far, all preimplantation genetic diagnosis (PGD) protocols in use produce results after the eggs have been fertilized. However, these approaches are not acceptable for patients with moral objections to the generation and discard of supernumerary zygotes or embryos. In these circumstances, only those oocytes to be replaced may be inseminated. The purpose of this study was to develop a PGD protocol to diagnose first polar bodies (PBs) prior to Intracytoplasmatic Sperm Injection (ICSI) in order to inseminate only those oocytes found to be chromosomally normal. PB biopsy was performed 1 hour after ovum pick up, and after fixation, the PBs were analysed by FISH and the eggs inseminated by ICSI no later than 7 hours after retrieval. One third (33.3%) of the PBs were aneuploid. Fifty-four normal and 12 non-resolved oocytes were injected by ICSI, of which 65% became 2-PN zygotes. Embryo transfer on day 2 was possible in all 10 patients (average maternal age 35.2+/-3.2, range 29-39 years), of which 6 became pregnant with 8 fetuses (28.6% or 8/28 transferred embryos). The results indicate that PB analysis of some common chromosome abnormalities is feasible within time limits imposed by ICSI insemination (6 hours or less)., (Copyright 2000 John Wiley & Sons, Ltd.)

In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins. Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP. This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays. Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.

During the first steps of the gamete interaction, the proacrosin/acrosin system seems to play a crucial role in the secondary binding, holding acrosome-reacted spermatozoa during their passage through the zona pellucida. To analyze the functional domains of acrosin, we decided to express recombinant boar acrosin proteins in bacteria and to study their binding capacities to zona pellucida glycoproteins (ZPGPs). The expressed proteins were immunodetected by Western blot with a polyclonal antiacrosin antibody. The recombinant truncated beta-acrosin has a typical hyperbolic curve of a zymogen enzymatic activation. Three of the five recombinant forms (truncated beta-acrosin, Ser/Ala222-truncated beta-acrosin, and truncated beta-acrosin "heavy chain") had the ability to bind ZPGPs. The two shorter forms (the amino and carboxy termini of truncated beta-acrosin) failed to bind. The catalytic site mutant (Ser/Ala222) of truncated beta-acrosin does not differ from the recombinant truncated beta-acrosin in its mechanism of interaction to ZPGPs, indicating that this secondary binding is done by a nonenzymatic process. Our results show that binding between acrosin and ZPGPs depends on the secondary and tertiary structures of acrosin and does not depend on an active catalytic site.

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95 kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, in in vitro fertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.

Mammalian sperm acrosomes contain a trypsin-like protease called acrosin which causes limited and specific hydrolysis of the extracellular matrix of the mammalian egg, the zona pellucida. Acrosin was localized on hamster, guinea-pig and human sperm using monoclonal and polyclonal antibodies to human acrosin labelled with colloidal gold. This was visualized directly with transmission electron microscopy, and with light and scanning microscopy after silver enhancement of the colloidal gold probe. Four distinct labelling patterns were found during capacitation and the acrosome reaction in hamster and guinea-pig spermatozoa, and three patterns were found in human spermatozoa. In the hamster, acrosin was not detected on the inner acrosomal surface after the completion of the acrosome reaction, thus correlating with the observation that hamster spermatozoa lose the ability to penetrate the zona after the acrosome reaction. With guinea-pig and human spermatozoa, acrosin was still detected after the completion of the acrosome reaction, thus correlating with the observation that acrosome reacted guinea-pig spermatozoa bind to and penetrate the zona pellucida.