Functional redundancy and compensation: Deletion of multiple murine Crisp genes reveals their essential role for male fertility.

Mammalian Cysteine-RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca2+ channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multiple Crisp gene mutants by CRISPR/Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2-/- .Crisp4-/- mice led to the generation of both triple and quadruple knockout mice exhibiting a complete or severe disruption of male fertility due to a combination of sperm transport, fertilization, and embryo developmental defects linked to intracellular Ca2+ dysregulation. These observations reveal that CRISP proteins are essential for male fertility and organize in functional modules that contribute distinctly to fertility success, bringing insights into the mechanisms underlying functional redundancy/compensation in protein families and emphasizing the importance of generating multiple and not just single knockout which might be masking the true functional relevance of family genes.

Relevance of CRISP proteins for epididymal physiology, fertilization, and fertility.

BACKGROUND: The molecular mechanisms involved in the acquisition of mammalian sperm fertilizing ability are still poorly understood, reflecting the complexity of this process. OBJECTIVES: In this review, we describe the role of Cysteine RIch Secretory Proteins (CRISP1-4) in different steps of the sperm journey to the egg as well as their relevance for fertilization and fertility. MATERIALS AND METHODS: We analyze bibliography reporting the phenotypes of CRISP KO mice models and combine this search with recent findings from our team. RESULTS: Generation of individual KO for CRISP proteins reveals they are key mediators in different stages of the fertilization process. However, in spite of their important functional roles, KO males for each of these proteins remain fertile, supporting the existence of compensatory mechanisms between homologous CRISP family members. The development of mice lacking epididymal CRISP1 and CRISP4 simultaneously (DKO) revealed that mutant males exhibit an impaired fertility due to deficiencies in the sperm ability to fertilize the eggs in vivo, consistent with the proposed roles of the two proteins in fertilization. Interestingly, DKO males show clear defects in both epididymal epithelium differentiation and luminal acidification known to be critical for sperm maturation and storage. Whereas in most of the cases, these epithelium defects seem to specifically affect the sperm fertilizing ability, some animals exhibit a disruption of the characteristic immune tolerance of the organ with clear signs of inflammation and sperm viability defects. DISCUSSION AND CONCLUSION: Altogether, these observations confirm the relevance of CRISP proteins for male fertility and contribute to a better understanding of the fine-tuning mechanisms underlying sperm maturation and immune tolerance within the epididymis. Moreover, considering the existence of a human epididymal protein functionally equivalent to rodent CRISP1 and CRISP4, DKO mice may represent an excellent model for studying human epididymal physiology and pathology.

Prevalence of intestinal parasitic infection among children from a shanty town in Tucuman, Argentina.

A parasitological survey was conducted in children living in a poor area next to a drainage channel, in Tucumán province, Argentina. Stool specimens from 115 children were collected and samples were analyzed by direct microscopy examination and concentration techniques. The prevalence rate of intestinal parasites infection was high (78.3%) and Blastocystis hominis was the most frequent protozoan parasite found (68.9%) followed by Giardia intestinalis (33.3%), Entamoeba coli (24.4%), Endolimax nana (12.2%), Chilomastix mesnili (5.6%) and Iodamoeba bütschlii (2.2%). Ascaris lumbricoides was the most prevalent intestinal helminth, with an infection frequency of 38.9%, followed by Trichuris trichiura (13.3%), Strongyloides stercoralis (13.3%), Hymenolepis nana (7.8%) and Enterobius vermicularis (3.3%). Multiple parasitic infections were also high, affecting 71.1% of infected population. These results indicate that sanitary policies, including health care and sanitary education have been inadequate for the control of intestinal parasitism in this high-risk population. Implementation of sanitation programs is a basic need and a joint collaboration between public servant and health professionals should be a priority.

Parasitological survey of schoolchildren from a highaltitude community from Tucumán Province, Argentina: Remarkable low detection of soil-transmitted helminths in comparison with coastal communities.

The prevalence of intestinal parasitic infections among schoolchildren in Colalao del Valle, a high-altitude community in Tucumán province, Argentina, was investigated. The data revealed a high prevalence of parasitism (79.7%) with no significant differences in distribution by sex or age. Protozoa infections were the most common with Blastocystis hominis being the most prevalent (62.5%), followed by Giardia lamblia (29.7%), Endolimax nana (15.6%), Entamoeba coli (12.5%) and Iodamoeba bütschlii (3.1%). Interestingly, there was an absence of soil-transmitted helminths among the studied population which could be related to climate (variable temperatures, moderate rainfall) and soil type (clay).

Probiotic administration modifies the milk fatty acid profile, intestinal morphology, and intestinal fatty acid profile of goats.

The effect of a mixture of potentially probiotic bacteria (MPPB; Lactobacillus reuteri DDL 19, Lactobacillus alimentarius DDL 48, Enterococcus faecium DDE 39, and Bifidobacterium bifidum strains) on the milk fatty acid (FA) profile, with emphasis on cis-9,trans-11 conjugated linoleic acid (CLA) in the middle stage of goat lactation, was determined. In addition, the effects of MPPB feeding on the FA profile in intestinal content and intestinal morphology in weaned goats were analyzed. The probiotic supplement was able to modify FA composition of milk and intestinal content. The unsaturated FA concentrations in milk (g of FA/L of milk) increased from 4.49 to 7.86 for oleic (18:1), from 0.70 to 1.39 for linoleic (18:2), from 0.063 to 0.187 for linolenic (18:3) acid, and from 0.093 to 0.232 for CLA. The atherogenicity index diminished 2-fold after MPPB ingestion. In the intestinal content of the weaned goats, no significant difference in saturated FA concentration compared with the control was observed. However, oleic acid, linolenic acid, CLA, and docosahexaenoic acid concentrations increased by 81, 23, 344, and 74%, respectively, after probiotic consumption. The ruminal production of CLA was increased by the MPPB. However, bacterial strains of MPPB were unable to produce CLA in culture media. By histological techniques, it was observed that the treated group had intestinally more conserved morphological structures than the control group. The results obtained in this study indicate that the MPPB administration in lactating and weaned goats allows for the production of milk with improved concentrations of beneficial compounds, and also produces a protective effect in the goat intestine. The results obtained in this study reinforce the strategy of probiotics application to enhance goat health with the production of milk with higher concentrations of polyunsaturated FA.

Parasitological survey of schoolchildren from a highaltitude community from Tucumán Province, Argentina: Remarkable low detection of soil-transmitted helminths in comparison with coastal communities

The prevalence of intestinal parasitic infections among schoolchildren in Colalao del Valle, a high-altitude community in Tucumán province, Argentina, was investigated. The data revealed a high prevalence of parasitism (79.7%) with no significant differences in distribution by sex or age. Protozoa infections were the most common with Blastocystis hominis being the most prevalent (62.5%), followed by Giardia lamblia (29.7%), Endolimax nana (15.6%), Entamoeba coli (12.5%) and Iodamoeba bütschlii (3.1%). Interestingly, there was an absence of soil-transmitted helminths among the studied population which could be related to climate (variable temperatures, moderate rainfall) and soil type (clay).

Prevalence of intestinal parasitic infection among children from a shanty town in Tucuman, Argentina

A parasitological survey was conducted in children living in a poor area next to a drainage channel, in Tucumán province, Argentina. Stool specimens from 115 children were collected and samples were analyzed by direct microscopy examination and concentration techniques. The prevalence rate of intestinal parasites infection was high (78.3%) and Blastocystis hominis was the most frequent protozoan parasite found (68.9%) followed by Giardia intestinalis (33.3%), Entamoeba coli (24.4%), Endolimax nana (12.2%), Chilomastix mesnili (5.6%) and Iodamoeba bütschlii (2.2%). Ascaris lumbricoides was the most prevalent intestinal helminth, with an infection frequency of 38.9%, followed by Trichuris trichiura (13.3%), Strongyloides stercoralis (13.3%), Hymenolepis nana (7.8%) and Enterobius vermicularis (3.3%). Multiple parasitic infections were also high, affecting 71.1% of infected population. These results indicate that sanitary policies, including health care and sanitary education have been inadequate for the control of intestinal parasitism in this high-risk population. Implementation of sanitation programs is a basic need and a joint collaboration between public servant and health professionals should be a priority.

Administration of Lactobacillus fermentum CRL1446 increases intestinal feruloyl esterase activity in mice.

AIMS: To evaluate the effect of oral administration of Lactobacillus fermentum CRL1446 on the intestinal feruloyl esterase (FE) activity and oxidative status of mice. METHODS AND RESULTS: Adult Swiss albino mice received Lact. fermentum CRL1446 at the doses 10(7) and 10(9) cells per day per mouse for 2, 5, 7 and 10 days. Intestinal FE activity, intestinal microbiota counts, plasmatic thiobarbituric acid-reactive substances (TBARS) percentage and glutathione reductase (GR) activity were determined. Mice that received Lact. fermentum CRL1446 at the dose 10(7) cells per day for 7 days showed a twofold increase in total intestinal FE activity, compared to the nontreated group. In large intestine content, FE activity increased up to 6·4 times. No major quantitative changes in colonic microbiota were observed in treated animals. Administration of this strain produced an approx. 30-40% decrease in the basal levels of plasmatic TBARS and an approx. twofold increase in GR activity from day 5 of feeding with both doses. CONCLUSIONS: Oral administration of Lact. fermentum CRL1446 to mice increases total intestinal FE activity, decreases the basal percentage of plasmatic lipoperoxides and increases GR activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus fermentum CRL1446 could be orally administered as a dietary supplement or functional food for increasing the intestinal FE activity to enhance the bioavailability of ferulic acid, thus improving oxidative status.

Inhibition of mycotoxin-producing Aspergillus nomius vsc 23 by lactic acid bacteria and Saccharomyces cerevisiae

The effect of different fermenting microorganisms on growth of a mycotoxin- producing Aspergillus nomius was assayed. Two lactic acid bacteria, Lactobacillus fermentum and Lactobacillus rhamnosus, and Saccharomyces cerevisiae, all of which are widely used in fermentation and preservation of food, were assayed on their fungus inhibitory properties. Assays were carried out by simultaneous inoculation of one of the possible inhibiting microorganisms and the fungus or subsequent inoculation of one of the microorganisms followed by the fungus. All three microorganisms assayed showed growth inhibition of the mycotoxin-producing Aspergillus strain. L. rhamnosus O236, isolated from sheep milk and selected for its technological properties, showed highest fungal inhibition of the microorganisms assayed. The use of antifungal LAB with excellent technological properties rather than chemical preservatives would enable the food industry to produce organic food without addition of chemical substances.

The burden of unscheduled health care for asthma in Latin America.

OBJECTIVES: To determine the level and cost of unscheduled health care resource use in adults and children across all asthma symptom severities in Latin America. METHODS: The level and cost of health care resource use were analysed for 2074 patients with asthma included in the Asthma Insights and Reality in Latin America (AIRLA) survey from 10 Latin American countries. Health care resource use was multiplied by country-specific unit costs to estimate average per-patient annual costs. Patients were classified as adults (> or = 16 years) or children (<16 years), with disease severity categorized using a symptom severity index. RESULTS: Persistent asthma symptoms were experienced by 53.1% of patients (50.1% of children and 54.6% of adults). In the year preceding the survey, 57.1% of patients required unscheduled health care resource use and 45.1% reported at least 1 emergency hospital contact. The percentage of patients reporting unscheduled health care resource use was greatest amongst those with severe persistent symptoms (71.9%) but it was also high in those with mild intermittent symptoms (45.7%). An average of 73.2% of annual costs of asthma-related health care for the 10 countries was due to unscheduled health care. Expenditure on unscheduled care was greatest amongst both adults and children with severe persistent asthma symptoms (US $558 and US $769, respectively). Adults and children with mild intermittent symptoms also incurred considerable unscheduled costs (US $204 and US $215, respectively). CONCLUSIONS: Poorly controlled asthma imposes a considerable cost burden driven by unscheduled health care resource use in Latin America. Treatments to control asthma and reduce the need for unscheduled health care could reduce this cost in both adults and children.

Inhibition of mycotoxin-producing Aspergillus nomius vsc 23 by lactic acid bacteria and Saccharomyces cerevisiae.

The effect of different fermenting microorganisms on growth of a mycotoxin- producing Aspergillus nomius was assayed. Two lactic acid bacteria, Lactobacillus fermentum and Lactobacillus rhamnosus, and Saccharomyces cerevisiae, all of which are widely used in fermentation and preservation of food, were assayed on their fungus inhibitory properties. Assays were carried out by simultaneous inoculation of one of the possible inhibiting microorganisms and the fungus or subsequent inoculation of one of the microorganisms followed by the fungus. All three microorganisms assayed showed growth inhibition of the mycotoxin-producing Aspergillus strain. L. rhamnosus O236, isolated from sheep milk and selected for its technological properties, showed highest fungal inhibition of the microorganisms assayed. The use of antifungal LAB with excellent technological properties rather than chemical preservatives would enable the food industry to produce organic food without addition of chemical substances.

Conjugated linoleic acid conversion by dairy bacteria cultured in MRS broth and buffalo milk.

AIMS: To evaluate strains of Lactobacilli, Bifidobacteria and Streptococci for their ability to produce conjugated linoleic acid (CLA) from free linoleic acid (LA). METHODS AND RESULTS: Eight dairy bacteria tolerant to LA were grown in MRS broth containing LA (200 microg ml(-1)) and CLA was assessed. Seven bacteria were able to form CLA after 24 h of incubation, varying percentage conversion between 17% and 36%. Lactobacillus casei, Lactobacillus rhamnosus, Bifidobacterium bifidum and Streptococcus thermophilus showed the highest LA conversion and were inoculated into buffalo milk supplemented with different concentration of LA. The production of CLA at 200 microg ml(-1) of LA was two- or threefold in milk than MRS broth. All evaluated strains were able to produce CLA from high LA levels (1000 microg ml(-1)). CONCLUSIONS: The most tolerant strain to LA was Lact. casei. Lacttobacillus rhamnosus produced the maximum level of CLA at high LA concentrations (800 microg ml(-1)). The selected bacteria may be considered as adjunct cultures to be included on dairy fermented products manufacture. Low concentration of LA must be added to the medium to enhance CLA formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of CLA by strains using milks from regional farms as medium offer a possible mechanism to enhance this beneficial compound in dairy products and those the possibility to develop functional foods.

Assessment of beta-glucuronidase levels in goat's milk as an indicator of mastitis: comparison with other mastitis detection methods.

The use of somatic cell counts (SCCs) for the diagnosis of mastitis is not a well-established procedure for the caprine species, because nonleucocytic cell-like particles are normally observed as a result of the apocrine secretion process of the goat mammary gland. The infection levels of 124 goats were measured by the beta-glucuronidase test, which was compared with the SCC method and the California mastitis test (CMT). Seventy-nine of 124 samples (63.7%) showed SCCs lower than 1.3 x 10(3) cells per ml. Of these samples, 93% showed low levels of beta-glucuronidase activity (< 15 U/ml). In the remaining 36.3% of the samples, SCCs were higher than 1.3 x 10(3) cells per ml. Of these samples, 88% showed high levels of beta-glucuronidase activity (15 to 100 U/ml). The CMT gave similar results. In this study, the beta-glucuronidase test was standardized for goat milk and shown to be reliable, enabling one to count only the somatic enzyme cells in milk and avoiding the interference encountered with the SCC method.

Cefuroxime vs a dicloxacillin/chloramphenicol combination for the treatment of parapneumonic pleural effusion and empyema in children.

The aim of this study was to evaluate the efficacy of cefuroxime, compared with the combination of dicloxacillin/chloramphenicol, for the treatment of children with parapneumonic pleural effusion or empyema. Forty patients, aged 3 months to 5 years, with pleural effusion or empyema were randomized to receive cefuroxime (100 mg/kg/day) IV (n=20) or chloramphenicol (100 mg/kg/day) plus dicloxacillin (200 mg/kg/day) IV (n=20). Both groups were similar in age, days of illness, clinical and radiological findings, and etiology. Most patients (70%) had an empyema at presentation. There was no difference in clinical outcomes, including days to defervescence, duration of respiratory distress, duration of chest tube drainage, and days to discharge from hospital. The complication rates were similar in both groups. Pleural thickening occurred in four patients, bronchopleural fistula in two, and loculated empyema in one patient of each treatment group. Adverse effects attributed to cefuroxime were mild and infrequent. These results suggest that cefuroxime is an effective and well-tolerated alternative for the treatment of children with pleural effusion and empyema.

Estudio multicéntrico de la eficacia y seguridad de amoxicilina/sulbactam en niños con otitis media aguda / Multicentric study of the amoxicillin/sulbactam effectiveness and safety in children with acute otitis media

Introducción. La otitis media aguda (OMA) es una de las principales causas de enfermedad en la infancia, y el motivo más común de visita al médico durante los primeros dos años de vida, después de las enfermedades virales de las vías respiratorias superiores. Los gérmenes causales de OMA pueden ser resistentes a los antibióticos utilizados para combatir esta enfermedad; es por ello que surge la necesidad de buscar nuevas alternativas terapéuticas. Material y métodos. Del 1 de agosto al 31 de octubre de 1999, en el Centro Médico Nacional (CMN) "20 de Noviembre" del ISSSTE, el Instituto Nacional de Pediatría (INP) de la Secretaría de Salud y el CMN "La Raza" del IMSS, de la ciudad de México, se realizó un estudio prospectivo en un grupo de 53 niños, de entre tres meses y seis años de edad, cuyo peso era menor de 25 kilogramos. En la primera visita se practicó examen clínico, otoscopia, cultivo de secreción ótica, y se les administró amoxicilina/sulbactam (Trifamox IBL 500) a dosis de 50 mg/kg/día cada 12 horas durante diez días. Posteriormente, se practicó nueva evaluación clínica en los días 1, 3, 10 y 40 después de iniciar el tratamiento para evaluar el cumplimiento, los efectos adversos y la eficacia del antibiótico. Resultados. De los 53 niños estudiados, 27 fueron del sexo femenino y 26 del masculino, con una edad promedio de 34.3 meses, un peso promedio de 14.9 kg y un error estándar de 1.1. Todos cumplieron con el tratamiento. A 30 niños se les practicó miringotomía con toma de secreción ótica, y los microorganismos aislados fueron: Streptococcus pneumoniae 36.7 por ciento (11/30), Haemophilus influenzae 33.3 por ciento (10/30), Staphylococcus aureus 3.3 por ciento (1/30), Staphylococcus epidermidis 3.3 por ciento (1/30), Klebsiella oxytoca 3.3 por ciento (1/30) y ninguno en 20.1 por ciento (6/30). Los efectos adversos fueron: ninguno en 18.9 por ciento (10/53), diarrea 13.2 por ciento (7/53), náusea 5.7 por ciento (3/53), rash 3.8 por ciento (2/53), dolor abdominal 1.9 por ciento (1/53), caída del cabello 1.9 por ciento (1/53) y dolor ocular en 1.9 por ciento (1/53). Se observó curación clínica en 98.1 por ciento de los niños.Conclusiones. La administración de amoxicilina/sulbactam como antibiótico es eficaz y segura en los niños que padecen OMA.

Study of adhesion of Lactobacillus casei CRL 431 to ileal intestinal cells of mice.

It is well known that the cell wall of Lactobacillus casei CRL 431, a strain present in probiotics, presents lectinlike surface molecules. Presence of these molecules stimulates the immune system. Given the role that lectins and lectinlike substances play in the adhesion phenomenon, it is probable that this is an initial stage in the immunostimulation produced by this bacterium. To confirm this, adhesion of this microorganism to exfoliated mouse ileal epithelial cells was studied in vitro. Other L. casei strains isolated from adult human intestines and one of dairy origin were also examined for their ability to adhere to ileal epithelial cells. Another strain, which was included in the present study, was Lactobacillus acidophilus CRL 730. L. casei strains isolated from humans showed good ability to adhere to ileal epithelial cells, whereas L. casei isolated from dairy origin did not. Adhesion was only observed at 37 degrees C and at a pH between 6 and 7.5. The exposure time needed for highest adhesion was 30 min. Presence of lectinlike substances on the surface of L. casei CRL 431 is important to this adhesion phenomenon, since adherence capacity was lost after removal of these substances.

Some probiotic properties of chicken lactobacilli.

The beneficial effect of lactobacilli has been attributed to their ability to colonize human and animal gastrointestinal tracts. In this work, adhesion assays with three lactobacillus strains and intestinal fragments obtained from chickens were assessed. Lactobacillus animalis and L. fermentum were able to adhere to three kinds of epithelial cells (crop, small and large intestines) with predominance to small intestine. Among the strains considered, L. fermentum subsp. cellobiosus showed the lowest and L. animalis the highest adhesion ability. Scanning electron microphotographs showing L. animalis and L. fermentum adhering to intestinal cells were obtained. The characterization of L. animalis adhesion indicated that lectin-like structure of this strain has glucose/mannose as specific sugars of binding. However, a calcium requirement was not observed. The adhesion of L. fermentum was reduced by addition of sialic acid or mannose (P < 0.01). These carbohydrates can be involved in the interaction between adhesin and epithelial surface. In this case, the dependence on bivalent cations was demonstrated. Lactobacillus fermentum was effective in reducing the attachment of Salmonella pullorum by 77%, while L. animalis was able to inhibit (90%, 88%, and 78%) the adhesion of S. pullorum, S. enteritidis, and S. gallinarum to host-specific epithelial fragments respectively. Our results from this in vitro model suggest that these lactobacilli are able to block the binding sites for Salmonella adhesion.

Biotherapeutic role of fermented milk.

Fermented milk was used as therapy in infantile diarrhoea due to post-gastroenteritis syndrome. This treatment eliminated the disease in 4.0 days (mean value, SD = 2.8; n = 13) and allowed patients to return to free feeding according to their age. The weight percentile variation during treatment with fermented milk (15 days) was higher in the patients showing 3rd degree malnutrition than in other children. Bacteriotherapy can restore faecal flora which has been lowered by diarrhoea. Our results showed that levels higher than 10(6) UFC lactobacilli/g of faeces correlated with a healthy status of the children. Clinical applications of fermented milk with a mixture of Lactobacillus casei and Lactobacillus acidophilus in the prevention of gastrointestinal disorders are possible.

In vitro studies on the growth of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The inhibitory effect of lactobacilli on growth of Shigella sonnei was studied. The effect was not due to pH alone, as addition of hydrochloric, lactic or acetic acids to culture media did not inhibit the normal growth of the shigellas. The degree of inhibition was measured by disc assay and showed that the inhibitory substance(s) can be extracellular and diffusible, varying the degrees of inhibition depending on the media tested. When broth was inoculated with mixed cultures of Lactobacillus and Shigella strains, the inhibition began at 6 h and the death phase at 9 h. The higher inhibition was produced by the mixture of lactobacilli (35.5 +/- 2.5% at 6 h culture, 57.4 +/- 1.9% at 9 h and 91.2 +/- 1.2% at 14 h). The degree of inhibition was higher when the relationship pathogen : lactobacilli was 1:10(3). The specific growth rate of lactobacilli and shigella was different in pure or mixed cultures. When the lactobacillus alone was grown for 12 h and the shigellas then added, the numbers of shigellas began to decrease immediately at 37 degrees C. This work shows that the Lactobacillus strains employed in fermented milk can be used to inhibit the growth of Sh. sonnei.

Inhibition of Shigella sonnei by Lactobacillus casei and Lact. acidophilus.

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei and Lact. acidophilus against Shigella sonnei was studied. There was a 100% survival rate in mice fed for 8 d with fermented milk and then dosed orally with Sh. sonnei. The survival rate in control mice was approximately 60% after 21 d. Colonization of the liver and spleen with Sh. sonnei was markedly inhibited by pretreatment with fermented milk. Differences in cell counts of 2-3 log units between treated and control mice were always obtained, shigellas were not detected in these organs by the 10th day in treated mice, while high levels were maintained in the controls. Higher levels of anti-shigella antibodies were found both in sera and in small intestinal fluid of mice treated with fermented milk, suggesting that the protective immunity could be mediated by the mucosal tissue. These results suggest that milk fermented with Lact. casei and Lact. acidophilus could be used as a prophylactic against gastrointestinal infections by shigellas.