Mitochondrial DNA content reduction in the most fertile spermatozoa is accompanied by increased mitochondrial DNA rearrangement.

STUDY QUESTION: Is there an association between male fertility and spermatozoa mitochondrial DNA (mtDNA) copy number and genome rearrangements? SUMMARY ANSWER: Normal spermatozoa not only have a lower mtDNA copy number but also more DNA rearrangements than spermatozoa of men with severe oligoasthenospermia (SOA). WHAT IS KNOWN ALREADY: While there is a consensus that mtDNA content is decreased in the most fertile spermatozoa, the role of mtDNA sequence alteration in male infertility is unclear. High-throughput sequencing, which allows an exhaustive analysis of mtDNA rearrangements and mutations, could be helpful in this context, but has yet to be used. STUDY DESIGN, SIZE, DURATION: This is an observational study of semen samples obtained from 44 men undergoing ART at an academic infertility centre in France, from October 2018 to November 2020. The men were classified into two groups: 20 men in the SOA group and 24 men with normal semen parameters in the control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: For each patient and control, mtDNA was isolated from sperm fractions from the 40% and 90% layers of the density gradient. The average mtDNA content of each sample was assessed using digital PCR. Deep sequencing was performed using next-generation sequencing. Signal processing and base calling were performed via the embedded pre-processing pipeline, the variants were analysed using an in-house workflow and a dedicated tool, based on soft-clipping, was used to study large mtDNA rearrangements. The distribution and the type of rearrangements and variants were compared between patients with SOA and controls on one hand, and between the 40% and 90% gradient layers, on the other hand. MAIN RESULTS AND THE ROLE OF CHANCE: The mtDNA content of spermatozoa in the SOA group was significantly higher than in the control group (P < 0.0001). Moreover, mtDNA content was significantly higher in spermatozoa from the 40% layer (the most fertile spermatozoa) compared to the 90% layer, both in the SOA (P = 0.02) and the control group (P < 0.0001). The frequency of large mtDNA deletions and duplications was significantly higher in the control group (P = 0.002). Most of these rearrangements are potentially related to DNA breaks and their number was reduced by the removal of the linear mtDNA from the samples. Heteroplasmic variants were found more frequently in the SOA group (P = 0.05) and in the 40% layer (P = 0.03), but none had any obvious functional consequence. LIMITATIONS, REASONS FOR CAUTION: Our findings are novel and significant but should be verified in larger cohorts and other types of male infertility. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest that sperm mtDNA rearrangements are not necessarily associated with mitochondrial dysfunction and male infertility. Instead, they seem to be concomitant with the process of mtDNA content reduction in the most potentially fertile spermatozoa. Furthermore, they refute the hypothesis that, in the case of mtDNA alteration, a compensatory mechanism allows an increase in mtDNA copy number to rectify the energy deficit. The increased frequency of mtDNA rearrangements in the most fertile spermatozoa is a novel result that offers new insight into the relation between sperm quality and mtDNA. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Angers University Hospital (grant AOI CHU Angers 2018), Angers University and the French national research centres INSERM and CNRS. There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Maternal ageing impairs mitochondrial DNA kinetics during early embryogenesis in mice.

STUDY QUESTION: Does ageing affect the kinetics of the mitochondrial pool during oogenesis and early embryogenesis? SUMMARY ANSWER: While we found no age-related change during oogenesis, the kinetics of mitochondrial DNA content and the expression of the factors involved in mitochondrial biogenesis appeared to be significantly altered during embryogenesis. WHAT IS KNOWN ALREADY: Oocyte mitochondria are necessary for embryonic development. The morphological and functional alterations of mitochondria, as well as the qualitative and quantitative mtDNA anomalies, observed during ovarian ageing may be responsible for the alteration of oocyte competence and embryonic development. STUDY DESIGN, SIZE, DURATION: The study, conducted from November 2016 to November 2017, used 40 mice aged 5-8 weeks and 45 mice aged 9-11 months (C57Bl6/CBA F(1)). A total of 488 immature oocytes, with a diameter ranging from 20 µm to more than 80 µm, were collected from ovaries, and 1088 mature oocytes or embryos at different developmental stages (two PN, one-cell, i.e. syngamy, two-cell, four-cell, eight-cell, morula and blastocyst) were obtained after ovarian stimulation and, for embryos, mating. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mitochondrial DNA was quantified by quantitative PCR. We used quantitative reverse transcriptase PCR (RT-PCR) (microfluidic method) to study the relative expression of three genes involved in the key steps of embryogenesis, i.e. embryonic genome activation (HSPA1) and differentiation (CDX2 and NANOG), two mtDNA genes (CYB and ND2) and five genes essential for mitochondrial biogenesis (PPARGC1A, NRF1, POLG, TFAM and PRKAA). The statistical analysis was based on mixed linear regression models applying a logistic link function (STATA v13.1 software), with values of P < 0.05 being considered significant. MAIN RESULTS AND THE ROLE OF CHANCE: During oogenesis, there was a significant increase in oocyte mtDNA content (P < 0.0001) without any difference between the two groups of mice (P = 0.73). During the first phase of embryogenesis, i.e. up to the two-cell stage, embryonic mtDNA decreased significantly in the aged mice (P < 0.0001), whereas it was stable for young mice (young/old difference P = 0.015). The second phase of embryogenesis, i.e. between the two-cell and eight-cell stages, was characterized by a decrease in embryonic mtDNA for young mice (P = 0.013) only (young/old difference P = 0.038). During the third phase, i.e. between the eight-cell and blastocyst stage, there was a significant increase in embryonic mtDNA content in young mice (P < 0.0001) but not found in aged mice (young/old difference P = 0.002). We also noted a faster expression of CDX2 and NANOG in the aged mice than in the young mice during the second (P = 0.007 and P = 0.02, respectively) and the third phase (P = 0.01 and P = 0.008, respectively) of embryogenesis. The expression of mitochondrial genes CYB and ND2 followed similar kinetics and was equivalent for both groups of mice, with a significant increase during the third phase (P < 0.01). Of the five genes involved in mitochondrial biogenesis, i.e. PPARGC1A, NRF1, POLG, TFAM and PRKAA, the expression of three genes decreased significantly during the first phase only in young mice (NRF1, P = 0.018; POLGA, P = 0.002; PRKAA, P = 0.010), with no subsequent difference compared to old mice. In conclusion, during early embryogenesis in the old mice, we suspect that the lack of a replicatory burst before the two-cell stage, associated with the early arrival at the minimum threshold value of mtDNA, together with the absence of an increase of mtDNA during the last phase, might potentially deregulate the key stages of early embryogenesis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Because of the ethical impossibility of working on a human, this study was conducted only on a murine model. As superovulation was used, we cannot totally exclude that the differences observed were, at least partially, influenced by differences in ovarian response between young and old mice. WIDER IMPLICATIONS OF THE FINDINGS: Our findings suggest a pathophysiological explanation for the link observed between mitochondria and the deterioration of oocyte quality and early embryonic development with age. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University of Angers, France, by the French national research centres INSERM and the CNRS and, in part, by PHASE Division, INRA. There are no competing interests.

The mitochondrial DNA content of cumulus cells may help predict embryo implantation.

PURPOSE: The quantification of mtDNA in cumulus granulosa cells (CGCs) surrounding an oocyte has been positively linked with morphological embryonic quality. In the present study, we evaluated the link between the amount of mtDNA in CGCs surrounding an oocyte and the chances for the corresponding embryo of implanting and leading to an ongoing pregnancy. METHODS: This is an observational study, performed on 84 oocyte-cumulus-complexes (OCCs) having led to the replacement of an embryo in the maternal uterus, retrieved from 71 patients undergoing IVF with intracytoplasmic sperm. The OCCs were classified in two groups, one including 26 OCCs having led to an implanted embryo and the other including 58 OCCs having led to a non-implanted embryo. The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique. RESULTS: Significantly higher mtDNA copy numbers in CGCs were associated with implanted embryos than with non-implanted embryos (mean 215 [sd 375] and 59 [sd 72], respectively; p < 104). Multivariate analysis, taking into account the women's age, the embryo quality, and the AMH level, suggests an independent relationship between the mtDNA content of CGCs and the potential of embryo implantation. CONCLUSION: During in vitro fertilization (IVF) procedures, the probability of the implantation of the embryo appears to be closely correlated to the mtDNA copy numbers in the CGCs. Our results highlight the interest of mtDNA quantification in GCGs as a biomarker of the potential of embryo implantation.

The mitochondrial DNA content of cumulus granulosa cells is linked to embryo quality.

STUDY QUESTION: Could the mitochondrial DNA (mtDNA) content of cumulus granulosa cells (CGCs) be related to oocyte competence? SUMMARY ANSWER: The quality of embryos obtained during IVF procedures appears to be linked to mtDNA copy numbers in the CGCs. WHAT IS KNOWN ALREADY: Oocyte quality is linked to oocyte mtDNA content in the human and other species, and the mtDNA copy number of the oocyte is related to that of the corresponding CGCs. Moreover, the quantification of CGC mtDNA has recently been proposed as a biomarker of embryo viability. STUDY DESIGN SIZE, DURATION: An observational study was performed on 452 oocyte-cumulus complexes retrieved from 62 patients undergoing ICSI at the ART Center of the University Hospital of Angers, France, from January to May 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: The average mtDNA content of CGCs was assessed by using a quantitative real-time PCR technique. The relationship between CGC mtDNA content and oocyte maturity and fertilizability, on one hand, and embryo quality, on the other, was investigated using univariate and multivariate generalized models with fixed and mixed effects. MAIN RESULTS AND THE ROLE OF CHANCE: No relationship was found between CGC mtDNA content and oocyte maturity or fertilizability. In contrast, there was a significant link between the content of mtDNA in CGCs surrounding an oocyte and the embryo quality, with significantly higher mtDNA copy numbers being associated with good quality embryos compared with fair or poor quality embryos [interquartile range, respectively, 738 (250-1228) and 342 (159-818); P = 0.006]. However, the indication provided by the quantification of CGC mtDNA concerning the eventuality of good embryo quality was seriously subject to patient effect (AUC = 0.806, 95%CI = 0.719-0.869). The quantity of CGC mtDNA was influenced by BMI and smoking. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The quantification of CGC mtDNA may indicate embryo quality. However, since it is affected by patient specificity, it should be used with caution. It remains to be seen whether this marker could directly predict the implantation capacity of the embryo, which is the main objective in IVF practice. WIDER IMPLICATIONS OF THE FINDINGS: Our study suggests that the quantification of CGC mtDNA may be a novel biomarker of embryo viability. However, patient specificity makes it impossible to establish a general threshold value, valid for all patients. Nevertheless, further studies are needed to determine whether the quantification of CGC mtDNA may, in combination with the morpho-kinetic method, offer an additional criterion for selecting the best embryo for transfer from a given cohort. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centres INSERM and the CNRS. There were no competing interests.

Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing.

STUDY QUESTION: Does ovarian ageing increase the number of heteroplasmic mitochondrial DNA (mtDNA) point mutations in oocytes? SUMMARY ANSWER: Our results suggest that oocytes are not subject to the accumulation of mtDNA point mutations during ovarian ageing. WHAT IS KNOWN ALREADY: Ageing is associated with the alteration of mtDNA integrity in various tissues. Primary oocytes, present in the ovary since embryonic life, may accumulate mtDNA mutations during the process of ovarian ageing. STUDY DESIGN, SIZE, DURATION: This was an observational study of 53 immature oocyte-cumulus complexes retrieved from 35 women undergoing IVF at the University Hospital of Angers, France, from March 2013 to March 2014. The women were classified in two groups, one including 19 women showing signs of ovarian ageing objectified by a diminished ovarian reserve (DOR), and the other, including 16 women with a normal ovarian reserve (NOR), which served as a control group. PARTICIPANTS/MATERIALS, SETTING, METHODS: mtDNA was extracted from isolated oocytes, and from their corresponding cumulus cells (CCs) considered as a somatic cell compartment. The average mtDNA content of each sample was assessed by using a quantitative real-time PCR technique. Deep sequencing was performed using the Ion Torrent Proton for Next-Generation Sequencing. Signal processing and base calling were done by the embedded pre-processing pipeline and the variants were analyzed using an in-house workflow. The distribution of the different variants between DOR and NOR patients, on one hand, and oocyte and CCs, on the other, was analyzed with the generalized mixed linear model to take into account the cluster of cells belonging to a given mother. MAIN RESULTS AND THE ROLE OF CHANCE: There were no significant differences between the numbers of mtDNA variants between the DOR and the NOR patients, either in the oocytes (P = 0.867) or in the surrounding CCs (P = 0.154). There were also no differences in terms of variants with potential functional consequences. De-novo mtDNA variants were found in 28% of the oocytes and in 66% of the CCs with the mean number of variants being significantly different (respectively 0.321, SD = 0.547 and 1.075, SD = 1.158) (P < 0.0001). Variants with a potential functional consequence were also overrepresented in CCs compared with oocytes (P = 0.0019). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Limitations may be due to the use of immature oocytes discarded during the assisted reproductive technology procedure, the small size of the sample, and the high-throughput sequencing technology that might not have detected heteroplasmy levels lower than 2%. WIDER IMPLICATIONS OF THE FINDINGS: The alteration of mtDNA integrity in oocytes during ovarian ageing is a recurring question to which our pilot study suggests a reassuring answer. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the University Hospital of Angers, the University of Angers, France, and the French national research centers, INSERM and the CNRS. There are nocompeting interests.

The Management of Persistent Distal Occlusions after Mechanical Thrombectomy and Thrombolysis: An Inter- and Intrarater Agreement Study.

BACKGROUND AND PURPOSE: The best management of patients with persistent distal occlusion after mechanical thrombectomy with or without IV thrombolysis remains unknown. We sought to evaluate the variability and agreement in decision-making for persistent distal occlusions. MATERIALS AND METHODS: A portfolio of 60 cases was sent to clinicians with varying backgrounds and experience. Responders were asked whether they considered conservative management or rescue therapy (stent retriever, aspiration, or intra-arterial thrombolytics) a treatment option as well as their willingness to enroll patients in a randomized trial. Agreement was assessed using κ statistics. RESULTS: The electronic survey was answered by 31 physicians (8 vascular neurologists and 23 interventional neuroradiologists). Decisions for rescue therapies were more frequent (n = 1116/1860, 60%) than for conservative management (n = 744/1860, 40%; P < .001). Interrater agreement regarding the final management decision was "slight" (κ = 0.12; 95% CI, 0.09-0.14) and did not improve when subgroups of clinicians were studied according to background, experience, and specialty or when cases were grouped according to the level of occlusion. On delayed re-questioning, 23 of 29 respondents (79.3%) disagreed with themselves on at least 20% of cases. Respondents were willing to offer trial participation in 1295 of 1860 (69.6%) cases. CONCLUSIONS: Individuals did not agree regarding the best management of patients with persistent distal occlusion after mechanical thrombectomy and IV thrombolysis. There is sufficient uncertainty to justify a dedicated randomized trial.

Early signaling events triggered by peroxovanadium [bpV(phen)] are insulin receptor kinase (IRK)-dependent: specificity of inhibition of IRK-associated protein tyrosine phosphatase(s) by bpV(phen).

Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) inhibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associated PTP whose inhibition by pVs correlates closely with IRK tyrosine phosphorylation, activation, and downstream signaling. pVs have also been shown to activate various tyrosine kinases (TKs) that could participate in activation of the insulin-signaling pathway. In the present study we have sought to determine whether pV-induced IRK tyrosine phosphorylation requires the intrinsic kinase activity of the IRK, and whether IRK activation is necessary to realize the early steps in the insulin-signaling cascade. To address this we evaluated the effect of a pure pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but not 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen) increased tyrosine phosphorylation and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transducing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185 and p60 with the src homology-2 (SH2) domains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3'-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in the insulin- signaling cascade follow from this activation event. This establishes a critical role for PTP(s) in the regulation of IRK activity. bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen) inhibits a PTP involved in the negative regulation of mitogen-activated protein kinases.

Specific inhibition by hGRB10zeta of insulin-induced glycogen synthase activation: evidence for a novel signaling pathway.

Grb10 is a member of a family of adapter proteins that binds to tyrosine-phosphorylated receptors including the insulin receptor kinase (IRK). In this study recombinant adenovirus was used to over-express hGrb10zeta, a new Grb10 isoform, in primary rat hepatocytes and the consequences for insulin signaling were evaluated. Over-expression of hGrb10zeta resulted in 50% inhibition of insulin-stimulated IRK autophosphorylation and activation. Analysis of downstream events showed that hGrb10zeta over-expression specifically inhibits insulin-stimulated glycogen synthase (GS) activity and glycogen synthesis without affecting insulin-induced IRS1/2 phosphorylation, PI3-kinase activation, insulin like growth factor binding protein-1 (IGFBP-1) mRNA expression, and ERK1/2 MAP kinase activity. The classical pathway from PI3-kinase through Akt-PKB/GSK-3 leading to GS activation by insulin was also not affected by hGrb10zeta over-expression. These results indicate that hGrb10zeta inhibits a novel and presently unidentified insulin signaling pathway leading to GS activation in liver.

Comparison of two methods in the determination of the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to acyclovir and alpha-interferon.

Two methods, the colorimetric method (neutral red dye uptake), and DNA hybridization using a HSV thymidine kinase gene probe (TK) have been used to examine the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to two antiviral drugs, acyclovir (ACV) and alpha-interferon (alpha-IFN). Using the colorimetric method, HSV isolates had ED50s ranging from 0.03 +/- 0.02 micrograms/ml to 0.164 +/- 0.03 micrograms/ml for ACV and 6.3 +/- 5.2 IU/ml to 55.0 +/- 11.4 IU/ml for alpha-IFN. With the DNA hybridization method, ED50s ranged from 0.033 +/- 0.012 micrograms/ml to 0.190 +/- 0.031 micrograms/ml for ACV and 8.5 +/- 5.0 IU/ml to 43.5 +/- 6.0 IU/ml for alpha-IFN. Two strains of HSV-1 were found to be resistant to very high concentrations of ACV (greater than 50.0 micrograms/ml). The values obtained by the two methods showed good correlation (r = 0.724, P = 0.002). Furthermore, our results demonstrate that the two methods are reproducible, reliable and the dye uptake assay is suitable for use in a diagnostic virology laboratory.

Microsatellite markers for genetic population studies in Glossina palpalis gambiensis (Diptera: Glossinidae).

Little is known about intraspecific variability in tsetse flies and its consequences for vectorial capacity. Microsatellite markers have been developed for Glossina palpalis gambiensis. Three loci have been identified and showed size polymorphisms for insectarium samples. G. palpalis gambiensis from Burkina Faso were also subjected to PCR to investigate then genetic variability. Amplifications were observed in different species belonging to the palpalis group. These molecular markers will be useful to estimate gene flow within G. palpalis gambiensis populations and analysis could be extended to related species.

Malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1: a clinicopathologic and molecular study of 17 patients.

OBJECTIVE: To identify potential prognostic factors and criteria for early detection of malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1 (NF1). DESIGN: Retrospective study of malignant peripheral nerve sheath tumors in a cohort of 395 patients with NF1 followed up between October 1, 1988, and January 1, 1999; review of the clinical and histological characteristics of treatment and course; and analysis of p53 mutations and overexpression in tumors. SETTING: Teaching hospital referral neurofibromatosis center for adults. PATIENTS: Seventeen patients with NF1 (9 males and 8 females). Mean +/- SD patient age at diagnosis was 32 +/- 14 years. MAIN OUTCOME MEASURES: (1) Clinical symptoms, (2) comparison of p53 mutations and overexpression in benign vs malignant tumors; and (3) median survival. RESULTS: Twelve patients had high-grade tumors. All tumors except 1 developed on preexisting nodular or plexiform neurofibromas. Pain and enlarging mass were the first and predominant signs. None of the benign tumors displayed significant p53 staining or p53 mutations. Six of 12 malignant tumors significantly overexpressed p53, and 4 of 6 harbored p53 missense mutations. Median survival was 18 months overall, 53 months in peripheral locations, and 21 months in axial locations. CONCLUSIONS: Malignant peripheral nerve sheath tumors are highly aggressive in NF1. They mostly arise from plexiform or nodular neurofibromas. Investigations and deep biopsy of painful and enlarging nodular or plexiform neurofibromas should be considered in patients with NF1. Late appearance of p53 mutations and overexpression precludes their use as predictive markers of malignant transformation.

Microsatellite markers for genetic population studies in Glossina palpalis (Diptera: Glossinidae).

Little is known about tsetse intraspecific variability and its consequences on vectorial capacity. Since isoenzyme analyses revealed little polymorphism, microsatellite markers have been developed for Glossina palpalis gambiensis species. Three loci have been identified and showed size polymorphisms for insectarium samples. Moreover, amplifications were observed in different species belonging to palpalis group. These molecular markers will be useful to estimate gene flow within G. p. gambiensis populations and analyses could be extended to related species.

New molecular marker for Trypanosoma (Duttonella) vivax identification.

Trypanosoma vivax is a widespread hemoparasite in tropical areas and is pathogenic to ruminant domestic livestock as well as wild ruminants. The accurate identification of parasites in both hosts and vectors is crucial for epidemiological studies and disease control programs. We describe here the development of molecular markers specific for T. vivax identification. These markers were used to identify mouthpart infections in field-collected tsetse flies from Cameroon. The markers target the genomic sequence of a species-specific antigen from the bloodstream stages. No cross amplification with other trypanosome species was observed, which makes the markers a reliable tool to detect T. vivax infections, both in hosts and vectors. The PCR-amplified sequence contains a (CA)(n) microsatellite repeat for which 11 different alleles were identified. This microsatellite, which showed high polymorphism, provides a suitable marker for population genetic studies.

Expression of basement membrane antigens and matrix metalloproteinases 2 and 9 in cutaneous basal and squamous cell carcinomas.

BACKGROUND: Basement membrane (BM) antigens and matrix metalloproteinases (MMP) are involved in tumor invasion and metastasis. Basal (BCC) and squamous cell carcinomas (SCC) differ with respect to their biological behavior since the former are only locally aggressive whereas the latter have a metastatic potential. MATERIALS AND METHODS: We studied the immunohistochemical expression of several BM antigens and of MMP2 and MMP9, in 13 BCC, 13 SCC, and 8 in situ skin carcinomas. RESULTS: The expression of most BM antigens was reduced in the tumors in comparison with normal skin. Hemidesmosome- and lamina lucida-associated antigens (plectin, NUT2, alpha 6/CD49f and laminin-5) were more decreased in BCC, whereas collagens type VII and IV were more decreased in SCC as compared with BCC; in BCC and SCC both collagens tended to be decreased on the leading edge of invasive tumor masses. In situ carcinomas showed a slightly diminished expression of alpha 6/CD49f integrin, plectin and NUT2. The expression of both MMP2 and MMP9 was increased in SCC as compared with BCC. CONCLUSION: Our findings further upheld the role of BM antigens and MMPs in the process of tumor aggressiveness. The reduced expression of collagen IV, combined with an increased expression of both MMP2 and MMP9 could account for the increased metastatic potential of SCC vs BCC through an increased invasion of the extracellular matrix and the vascular space.

Induction of tolerance by administration of hapten-immunoglobulin conjugates is associated with decreased IL-2 and IL-4 production.

Intravenous administration of trinitrophenyl-modified isologous immunoglobulin-induced nonresponsiveness to subsequent epicutaneous painting of sensitizing doses of trinitrochlorobenzene. Isologous immunoglobulin with various degrees of trinitrophenyl substitution (11.2, 14.3, 27 and 47.3) prevented sensitization. The suppression of contact hypersensitivity was dependent on the dose of tolerogen and was hapten specific. Tolerance was inducible in mice of the strains CBA (H-2k), C57BL/6 (H-2b), and DBA/2 (H-2d) but not in Balb/C (H-2d) mice, suggesting that this trait maps outside the murine major histocompatibility complex. Tolerance induced by trinitrophenyl-modified immunoglobulin was associated with decreased hapten-induced proliferation of draining lymph-node cells. Unlike in other models of tolerance in which a decreased interleukin-2 to interleukin-4 ratio can be observed, administration of tolerizing trinitrophenylated immunoglobulin was associated with deficient hapten-induced release of both interleukin-2 and interleukin-4.

Improvement of blood and fly gut processing for PCR diagnosis of trypanosomosis.

We have adapted a simple and efficient technique to detect trypanosomes in human blood, without DNA purification, and increased the sensitivity threshold to 1 parasite in 1 ml. We have then applied it for detection of parasites in midguts of tsetse flies, negative by microscopy. This technique has been developed for field conditions and could greatly facilitate epidemiological studies.

[Perianal streptococcal dermatitis]. / Anite périanale streptococcique.

BACKGROUND: Pediatric perianal streptococcal dermatitis (PSD) is a well-defined clinical entity. However, its highly uniform presentation remains surprisingly unrecognized by many practitioners 33 years after its first description. CASE REPORT: A seven-year-old girl had a three-week history of perianal and vulva redness with well-defined margins. Functional symptoms associated perirectal tenderness and pain during defecation, which was responsible for constipation. At onset she also presented with a sore throat, which resolved spontaneously, and she had been complaining for a few days about a perioral impetigo. She received mycostatin unsuccessfully for an alleged candidiasis. Positive cultures for group A beta-hemolytic streptococci from both perirectal and perioral swabs confirmed the diagnosis of PSD. Therapy with amoxicillin (50 mg/kg/d) was prescribed for ten days. Perianal lesions were cleared by day 2. CONCLUSION: Since PSD can masquerade as candidiasis, psoriasis, seborrheic dermatitis, inflammatory bowel disease or even sexual abuse, it remains an underdiagnosed entity. This situation leads to delayed diagnosis and treatment which in turn might increase the frequency of secondary complications related to streptococcal infections (i.e., post-streptococcal acute nephritis and rheumatism, guttate psoriasis, etc.).