Hemophagocytic lymphohistiocytosis (HLH) is a rare non-neoplastic, frequently fatal disease of childhood. It has been divided into two categories, familial and secondary, but the distinction between the forms is not easily made2. J.I. Henter, G. Elinder and A. Ost, FHL Study Group of the Histiocyte Society. Diagnostic guidelines for hemophagocytic lymphohistiocytosis. Semin Oncol 18 (1991), p. 29. View Record in Scopus | Cited By in Scopus (389). Clinical features including high fever, hepatosplenomegaly, and several neurological manifestations associated with pancytopenia, hypertriglyceridemia, hypofibrinogenemia, elevated liver enzymes, and hyperferritinemia are seen mainly in small children. HLH is considered a defect in immunomodulation, characterized by a benign, generalized lymphocytic and histiocytic proliferation associated with hypercytokinemia. The use of etoposide, steroids, and intrathecal methotrexate has led to remission in some patients; nevertheless, most patients still relapse and ultimately die from the disease. HLA-matched bone marrow transplantation (BMT) can bring about long-term remission. In the majority of patients, the transplant conditioning regimen consisted of busulfan, cyclophosphamide, and etoposide, which was well tolerated but accompanied by relevant toxicity, mainly severe oral mucositis. We report the beneficial effect of BMT in one patient, using a less toxic conditioning regimen.

A 2-month-old male, the third child of unrelated parents, was admitted with a history of 7 days of fever, abdominal distention, and jaundice. The physical examination showed an enlarged liver and spleen, 6 and 4 cm below the costal margin, respectively. Hemoglobin concentration was 78 g/L, total neutrophils 0.75 × 10⁹/ L, and the platelet count, 91 × 10⁹/L. Increases in direct bilirubin (82 ?mol/L), lactic dehydrogenase (660 IU), triglycerides (3.3 mmol/L), and decreased fibrinogen of 1.16 g/L were reported. The search for syphilis, toxoplasma, rubella, herpes, cytomegalovirus, and Epstein Barr virus was negative. The cerebrospinal fluid showed 15 mononuclear cells and hemophagocytosis was detected in the bone marrow, establishing the diagnosis of hemophagocytic lymphohistiocytosis.

The patient was given etoposide (150 mg/m²/day) for 3 consecutive days and dexamethasone (0.3 mg/kg/day) on 7 consecutive days, in addition to intrathecal methotrexate. Four weeks later, the patient was in complete remission, the physical examination was normal, and all of the altered laboratory studies had returned to normal limits. Cyclosporine at 5 mg/kg/day was initiated.

Three months later, allogenic BMT was performed. The donor was the patient’s healthy 8-year-old, HLA-identically-matched sister. The conditioning regimen included busulfan (4 mg/kg/days ?8, ?7, ?6, and ?5), etoposide (300 mg/m²/day on days ?3 and ?2), and cyclophosphamide (50 mg/kg/day on days ?4, ?3, and ?2). The number of nucleated cells infused was 4 × 10⁸/kg body weight of the recipient. Prophylactics for graft-vs.-host disease included methotrexate (10 mg/m² on days +3, +5, and +11), and cyclosporine 2 mg/kg twice per day i.v. Intravenous gamma globulin (65 mg/kg every 3 weeks until day +100) was used. An absolute neutrophil count of 500/uL was noted on day +12, the last day of platelet transfusion was day +14, and the patient was discharged on day +16.

Manifestations of GVHD in skin and liver were noticed on day +80 and required oral prednisone for its control. At present (day +400), the patient is asymptomatic, his physical examination is normal, and slightly increased GGT and alkaline phosphatase are the only laboratory abnormalities.

In the treatment of HLH, long-term remissions have been obtained with the use of systemic and intrathecal chemotherapy. However, most children treated with etoposide and steroids eventually relapse and die of the disease. Therefore, the use of allogeneic BMT appears to be a logical approach to the treatment of HLH regardless of whether excessive macrophage activation is intrinsic or secondary to T-cell activation. A recent report from the International HLH Registry cited an estimated 5-year survival rate of 68% after matched sibling transplantation, compared with 10.1% for patients treated solely with chemotherapy (p = 0.0001).

In the majority of patients, the transplant conditioning regimen has consisted of oral busulfan at 4 mg/kg/day (total dose 16 mg/kg), intravenous cyclophosphamide at 50 mg/kg/day (total dose 200 mg/kg), and intravenous etoposide at 500 mg/m²/day (total dose 1,500 mg/m²). This regimen has been well tolerated, but relevant toxicity, mainly severe oral mucositis has been reported without excessive incidence of transplant-related mortality. Some patients have also received intravenous ATG and aracytine. In some cases, toxicity was reduced by decreasing the etoposide dose from 1,500–900 mg/m² (300 mg/m²/day). The case reported here, the transplant conditioning regimen consisted of the same dose of oral busulfan, but reduced doses of cyclophosphamide (150 mg/kg) and etoposide (600 mg/m²). This regimen was adequate for the eradication of the disease and allowed persistent engraftment without significant toxicity. High doses of busulfan associated with cyclophosphamide have strong myeloablative and immunosuppressive effects. The inclusion of etoposide provides a third drug known to be effective in the treatment of HLH.

It is not always necessary to administer very high and potentially toxic doses of chemotherapy to eradicate the disease. In adults with leukemia, the use of cyclophosphamide at a dose of 120 mg/kg instead of 200 mg/kg did not compromise the antileukemic activity of the original regimen and markedly reduced the complication rate. Recently, the use of non-myeloablative stem cell transplantation has been successfully attempted as an alternative to conventional BMT with lethal cytoreduction for the treatment of malignant and non-malignant hematological diseases. The results in our patient suggest that a less toxic regimen permits a rapid engraftment of the donor stem cells without compromising the effectiveness of the chemotherapy. Fewer myeloablative or non-ablative regimens may also help bypass frequent late complications that result from the combined effects of high doses of chemotherapy in addition to prior conventional treatments. In the low-age group, allogeneic minus myeloablative BMT may reduce the incidence of growth retardation and infertility due to the unique sensitivity to chemoradiotherapy of the growth centers of the bones, gonads, and testicles. The use of non-myeloablative BMT should be attempted in HLH to reduce the morbidity and mortality related to this modality of treatment.

At the beginning of the century, urban rabies was adequately controlled in European countries through the use of vaccines obtained from the nervous tissue of various animal species infected with the rabies virus (Pasteur, Fermi, Semple). However, these vaccines contained myelin and other foreign protein residues and frequently produced neurological-type adverse reactions.

In 1956, Fuenzalida and Palacios developed a vaccine produced in suckling mouse brain, dramatically reducing the myelin content, but not the presence of other proteins foreign to humans. Later, the duck embryo vaccine, whose side effects decreased at the expense of immunogenicity, was produced. Finally, in the 1970s vaccines produced for human use in cell cultures were developed that in addition to preserving immunogenicity, eliminated the majority of the adverse effects produced by the myelin content and other foreign proteins. The VERO cell vaccine and the human diploid cell vaccine belong to this group.

The cell line used in the production of the latter vaccine is the diploid cell MRC-5, which avoids the appearance of undesirable mutations. It is, therefore, not considered as oncogenic, differing from vaccines used in animals. However, there should be continued periodic testing on vaccines produced in these cells to confirm the absence of their oncogenic ability.

The prevention of human rabies in Mexico continues to be an important goal for the health sector. Although the rate of human rabies has fallen significantly during the last 2 years, there are still cases of human rabies registered. Between 1990 and 1995, a total of 238 cases of human rabies were registered (an average of 40 cases annually), with a mean annual incidence of 0.04 cases per 100,000 inhabitants and a mortality rate of almost 100%. During 1998, 15 cases of human rabies were reported in Mexico, for a rate of 0.012 cases per 100,000 inhabitants, exhibiting an important decrease when compared to previous yearly rates of 0.02 (1996) and 0.02 (1997).

Based on data from the Panamerican Center for Zoonosis (CEPANZO) from 1985 to 1989, Mexico and Brazil were the countries registering the greatest number of deaths due to human rabies (rates of 0.092 and 0.03 per 100,000 inhabitants, respectively).

The official epidemiologic data for Mexico has annually reported between 70,000 and 80,000 persons hurt by animals potentially infected with the rabies virus, of whom approximately 30,000 receive vaccinations. In taking this into consideration, it is important to have highly effective vaccines with minimal side effects.

Side effects were generally much more troublesome with vaccine obtained from nervous tissue of animals than vaccine produced in cell cultures. Nevertheless, some signs related to human diploid cell antirabies vaccine consisting of urticarial rash and fever have been reported.

The objectives set forth for this study were to evaluate seroconversion to the human diploid cell antirabies vaccine through the measurement of vaccine-induced antibody levels achieved by subjects exposed to rabies who received or did not receive antirabies hyperimmune gamma globulin, to ascertain tolerance to this vaccine by measuring the type and frequency of adverse reactions after its application.

This is a comparative transverse study carried out between December 1, 1989 and June 30, 1995 in two tertiary-level pediatric hospitals located in Mexico City.

Forty children and adults were included with diverse histories of exposure to animals suspected of having rabies. These persons requested the antirabies vaccine application from the Preventive Medicine Department at one of the hospitals mentioned previously. Some patients came from other second-level hospitals, from health centers, or from direct physician referrals from the private sector. The administration of human hyperimmune gamma globulin was used following the criteria for a severe exposure. Hyperimmune gamma globulin was not, however, administered to all severe cases because on some occasions the product was not obtainable.

All subjects treated for post-exposure prophylaxis after contact with animals positive for rabies or suspected of having the disease were included. All subjects previously vaccinated with any other type of rabies vaccine or those who were immunosuppressed were excluded.

All patients received a complete scheme (five doses) of the human diploid cell vaccine, applied i.m. on days 0, 3, 7, 14, and 28 post-exposure. All of the previously mentioned treatments were performed after obtaining written informed consent from the patient or legal guardian.

Initially, general identification data were recorded for each patient and an interview was conducted 3 days after the application of each dose in order to assess the presence of any adverse post-vaccine effects, including induration, erythema, heat, pain at the application site, itching, fever, headaches, regional adenopathy, or anaphylactic reaction.

Two blood samples were taken (at baseline and at the end of the vaccination scheme). Antibody titers against the rabies virus were measured using a commercial ELISA diagnostic kit (Platelia r rage, Pasteur Diagnostic, Paris, France). This kit is an immuno-enzyme technique for the detection of rabies virus antiglycoprotein antibodies in human serum or plasma. The test is based on the use of a solid phase prepared with the glycoprotein extracted from the inactivated, purified virus membrane and an enzymatic conjugate (protein A from Staphylococcus aureus coupled with peroxidase). Comparison with a control serum titrated in international units IU/mL provided the titer of the unknown serum in equivalent units (the unit equivalent to the international units defined by seroneutralization).

Written authorization and consent were requested from each participating patient. Based on previously mentioned data, a specific format was designed in which the central objective of the study was explained, including data on the type of biological information to be used, days and dates of application, number of doses, and the potential side effects in the taking of blood samples—one at baseline (previous to the beginning of the vaccination scheme) and a second at the end. The patient’s signature or that of the parent or guardian, in the cases of minors, was included in addition to the principal investigator’s signature.

Simple frequencies, central tendency, and dispersion measures were estimated. For the comparison between the occurrence of adverse effects produced by the diploid cell vaccine and that reported in the literature for the Fuenzalida vaccine, a Z approximation to the binomial distribution was used, in addition to estimating the p value. Data are shown in tables and graphs, as well as on a histogram.

A total of 40 patients were included, 12 adults (30%) and 28 minors (70%), with an average age of 12.7 years (SD 11.1 years). The time between the exposure and the application of the first vaccine dose ranged between 1 and 15 days, with an average of 3.9 days.

Methicillin resistance in both Staphylococcus aureus and coagulase-negative staphylococci (CNS) has been extensively studied over the past three decades. Methicillin-resistant S. aureus (MRSA) was first recognized as a condition of poor clinical significance before observing a sudden increase in European countries by 1968. In the United States, the first hospital outbreak of MRSA was reported in 1968, and an epidemic rate was not observed until 1981. Later, many outbreaks were reported from Africa, Australia, and Asia in 1974, 1987, and 1988, respectively. Once MRSA has been introduced into a hospital, it may cause more than 50% of the institution’s hospital-acquired staphylococcal infections, usually difficult to eradicate.

Methicillin resistance in staphylococci is due to the presence of penicillin-binding protein 2a that exhibits low affinity for ?-lactam antibiotics. This protein is encoded by the mecA gene, located on the chromosome of all MRSA. While this gene encodes for the production of penicillin-binding protein 2a, auxiliary genes such as femA and femB encode proteins that influence the level of methicillin resistance, defining this phenotype as either heterogeneous, which is more prevalent, or as homogeneous. This highly epidemic organism also shows a multi-drug resistance profile, in which an association between methicillin and aminoglycoside resistance is common.

CNS has been less extensively studied than MRSA, but it has become a significant nosocomial pathogen, principally among infections related to medical devices. In clinical practice, S. epidermidis and S. hemolyticus have become the most significant species of CNS among methicillin-resistant CNS. In contrast to MRSA, only 81.5% of S. epidermidis and 58.3% of other CNS carry mecA. However, they do not have femA or femB genes, although S. epidermidis is the more common species and S. hemolyticus has become a meaningful organism because of its ability to develop vancomycin resistance, causing severe nosocomial infections. A possible pathogenic role of other biological properties, such as synergistic hemolysis and slime production, has been studied in CNS, but the association of such properties to methicillin resistance has not been thoroughly explored. Therefore, we performed a laboratory-based case-control study to determine the rate of infections caused by either MRSA or MRCNS. Additionally, we studied the relationship of methicillin resistance with synergistic hemolysis and the slime production of such microorganisms.

A laboratory-based survey was done at a 180-bed tertiary-care center in Mexico City. The surveillance method included a daily visit to the Clinical Microbiology Laboratory to review all inpatient records compiling staphylococci isolates. Whenever this occurred, an investigator interviewed the patient and checked the patient’s ward chart to register the following data: age; sex; underlying disease; antimicrobials used during the previous 4 weeks; length of hospital stay, and clinical manifestations of infection. All consecutive inpatients with a staphylococcal infection were included from May 1991 to October 1992. A non-matched case-control study was performed selecting two controls [patients with hospital-acquired methicillin-susceptible staphylococcal (MSS) infections] per case [patient with methicillin-resistant staphylococcal (MRS) infection].

All infections clinically apparent or in the incubation period at hospital admission were termed community-acquired infections. Nosocomial infection was acquired after a 72-h hospitalization or related to an intervention during the hospital stay. Staphylococcal infections were defined as follows: urinary-tract infection by midstream sample showing >10⁴ CFU/mL or ?10² CFU/mL in urine obtained by needle aspiration of a vesical catheter; intravenous catheter-related infection by detection of > 15 CFU isolated from a catheter segment (2 inches); wound infection by isolation of staphylococcus as the predominant organism in wound secretion; bacteremia by evidence of CNS in at least two blood cultures obtained from different anatomic sites, and for S. aureus, at least one positive blood culture. An episode of infection was defined as symptomatic or asymptomatic by the presence or absence of either clinical symptoms or physical signs.

S. aureus was identified by coagulase production. Species of CNS were defined by means of a commercial micromethod (UniScept 20GP, Analytab-Products, Plainview, NY, USA).

Screening of methicillin resistance was done by inoculating a 10-?L aliquot of a bacterial suspension with 3 × 10⁶ CFU/mL (McFarland turbidity standard no. 1) on Müeller-Hinton agar supplemented with Ca (50 mg/L), Mg (25 mg/L), NaCl (4%), and oxacillin (4 ?g/mL). All growth of at least one well-defined colony on these plates incubated at 30°C for 48 h was suggestive of MRS.

The methicillin-resistance trait was confirmed by microdilution in Müeller-Hinton broth supplemented with cations and NaCl, using a final inoculum of 7.5 × 10⁴ CFU. MRS were microorganisms with a minimal inhibitory concentration (MIC) of oxacillin ?8 ?g/mL, and those with a MIC ?1 ?g/mL were considered MSS. An MIC of oxacillin between ?1 ?g/mL and <8 ?g/mL defined a methicillin-intermediate staphylococcus (MIS). MICs for vancomycin, gentamicin, erythromycin, penicillin, tetracycline, clindamycin, amikacin, ciprofloxacin, and trimethoprim-sulfamethoxazole were carried out according to the recommendations of the National Committee for Clinical Laboratory Standards, using S. aureus ATCC 29213 as control.

Heterogeneous or homogeneous phenotype of MRS were defined according to the efficiency of plating (EOP) defined by the index: % = number of CFU on Müeller-Hinton agar supplemented with 50 ?g/mL of oxacillin divided by the number of CFU on regular Müeller-Hinton agar after incubation at 35°C for 96 h. An EOP index <1% or ?1% determined a phenotype as heterogeneous or homogeneous, respectively.

All clinical isolates were tested for synergistic hemolysis on trypticase soy agar plates containing 5% defibrinated sheep, ox, or human blood cells. S. aureus ATCC 25923, a well-known ?-lysine producer, was streaked perpendicularly to, but not touching, the testing organisms on each of the three plates and incubated at 35°C for 48 h. A positive test was defined as the presence of complete hemolysis observed in the area of partial hemolysis caused by S. aureus ATCC 25923 in either of the three plates.

All CNS were tested for slime production by a qualitative method, inoculating the organisms in a glass tube with trypticase soy broth. After incubation at 35°C for 48 h, the broth was poured off, and the presence of a film on the walls identified an isolate as a slime producer.

Forty-three cases and 86 controls were calculated as the size needed based on: (a) exposure rate to prolonged hospital stay (? days) of 0.20; (b) value of risk associated to exposure equal to or greater than 3; (c) -value of 0.05 (one-sided); and (d) ?-value of 0.20 (one-sided). An association between the epidemiologic characteristics of the population and MRS infection (dependent-variable) was explored by multiple logistic regression analysis [Epidemiological Graphics Estimation and Testing (EGRET) Cytel Software Corporation; Cambridge, MA, USA] of the variables that showed a p value of less than 0.10 in the univariate evaluation. Either the ?² test with Yates’ correction or Fisher’s exact test was used to compare proportions as required by using True Epistat Software (Epistat Services, Richardson, TX, USA). The relationship among biological traits was estimated by the Kappa index, in which one was considered as perfect association while zero (0) suggested correlation by chance. A p value of less than 0.05 was interpreted as significant.

Huntington’s disease (HD) is a late-onset, progressive neurodegenerative disorder. It has an autosomal dominant inheritance pattern and is characterized by involuntary movements, psychiatric features, and cognitive deterioration. Symptoms usually present at 37 to 45 years of age and the disease has a fatal outcome 15 to 20 years later. European populations have a reported frequency of 1 in 10,000. In 1983, Gusella et al. localized the gene to the short arm of chromosome 4 and the gene was cloned in 1993, finding a CAG triplet which repeats 6 to 29 times in the normal population and 36 to 120 times in affected individuals. Several countries have had protocols for predictive and prenatal diagnosis of the disease since 1986. This disease has become a model for the study of the ethical, moral and social problems predictive testing entails.

More neurodegenerative disorders are discovered daily that share many similar characteristics with HD (late onset, progressive, disabling, and no preventive or curative therapy) such as ataxias (types 1, 2, and 3) and familial Alzheimer’s disease. In some of these, predictive diagnosis is also available.

The purpose of this study was to explore the knowledge and attitudes regarding the disease and its predictive and prenatal diagnosis of a group of Mexican specialists, who because of their medical specialty might come into contact with HD patients.

A self-administered, 30-item multiple-choice questionnaire was completed by 147 professionals. The questionnaire included sociodemographic data, questions regarding HD knowledge and previous experience with HD, opinions on predictive and prenatal diagnosis, and views regarding social assistance for patients and their families. Some questions were adopted from the Thies et al. survey prepared in Germany.

The distributions of continuous variables are described with means and standard deviations, in addition to categorical variables with simple percentages. To compare the distribution of each variable codified categorically among the different professionals (neurologists vs. psychiatrists vs. psychologists), we used the statistic Pearson’s ?². This statistical test was additionally used for comparing differences among categorical variables codified in a binary manner with Yates’ correction or Fisher’s exact test as appropriate. The alpha value was set at the 0.05 level.

Of the 147 professionals, 62 were neurologists, 43 psychiatrists, 39 psychologists, and 3 did not state their profession. Forty percent of the professionals worked in government institutions, 17% in private practice, 32% in both, 3% in universities, and 10% in more than two places. Mean age was 35 years (±8.12 SD) with a range of 21–65 years. Gender distribution was 65 (44%) female and 82 (56%) male. Seventy-eight percent of the professionals were religiously affiliated, 21% were not, and 1% did not reply.

Only 11 subjects (8%) answered positively in regard to providing genetic counseling. One hundred eighteen professionals (80%) preferred to refer couples to the genetics unit, 9 (6%) were ignorant of how to deal with the couples, 1 (0.6%) did not supply any information, and 8 (5%) did not reply. There was no statistically significant difference for this parameter among the different professional groups. Neurologists provided information with the highest frequency (64%), followed by psychiatrists (25%), and psychologists (11%).

One hundred five (74%) professionals admitted to knowledge on the availability of predictive diagnosis through DNA testing. Fifty-one percent were neurologists, 28%, psychiatrists, and 21%, psychologists, with a statistically significant difference among the groups (?² 13.47, p <0.001). Only 10% of participants were aware of the World Neurology Federation guidelines for HD protocols.

Results regarding the recommendation of carrying out a test for a person at risk for the disease. One hundred twenty-seven professionals would encourage the predictive test, of whom 55 were neurologists, 38 psychiatrists, and 34 psychologists. There was no statistically significant difference among the groups. When questioned about the reasons for recommending the test, 81 (55%) replied that they would only recommend it if the subject was considering having offspring, 61 (41%) would recommend it taking into consideration the patient’s professional reasons, 9 (6%) would recommend it only if a treatment existed, and 18 (12%) did not reply. No statistically significant difference was found among the groups of specialists (this question had more than one option for response).

A total of 57 neurologists, 34 psychiatrists, and 35 psychologists believed that predictive diagnosis must be carried out only in laboratories collaborating with a genetics unit providing back-up by neurologists, psychiatrists, and psychologists. Five neurologists, 6 psychiatrists, and 2 psychologists considered this unnecessary; 1 psychiatrist and 2 psychologists did not know, and 5 did not reply.

One hundred forty professionals considered that the results of predictive diagnosis must be disclosed under the supervision of a neurologist, a psychiatrist or psychologist, and a genetic counselor, while 4 replied that it was not necessary, 1 did not know, and 2 did not reply.

With a positive test, indicating that the person will develop the disease, subjects were asked about the influence it would have on the patient’s decision to have offspring. When questioned about prenatal diagnosis for a patient with HD, 53 neurologists, 38 psychiatrists, and 33 psychologists answered that they recommended it. Five neurologists, 2 psychiatrists, and 2 psychologists replied no, 4 neurologists, 3 psychiatrists, and 2 psychologists replied that they did not know, and 5 had no reply. Among those whose reply was affirmative 56 were females and 68 males. Ninety-seven professionals were religiously affiliated and 27 were not. Among those who answered “no”, 2 were female and 7 were male. Seven were religiously affiliated and two were not. Of those who responded that they did not know, 5 were female and 6 were male, among whom 9 were religiously affiliated.

This study shows that the group we surveyed does not have adequate knowledge of HD. Only 59% of the subjects stated that they knew about the inheritance risks of the disease, 20% of whom had an incorrect concept. The study demonstrated that most of the professionals (80%) chose to refer patients to a specialized unit and therefore not provide genetic counseling.

Although a large number of subjects (74%) knew of the possibility of predictive diagnosis, only 10% were aware of the recommended guidelines for HD programs of the World Federation of Neurology HD working group and the HD International Association, in spite of the fact that they were published in a journal widely distributed among mainly neurologists. This might explain the general willingness of the respondents to cooperate with a genetic unit instead of providing counseling HD themselves. One noteworthy fact is that 74% of the subjects acknowledged the possibility of predictive diagnosis for HD, yet only 55% had previous contact with HD patients or were familiar with the disease.

Cyclosporine is widely used as an immunosuppressive agent, especially in organ transplantation. Since its introduction, kidney graft survival has considerably improved. However, cyclosporine also produces serious side effects, such as nephrotoxicity, hypertension, and liver and cerebral toxicity. However, the wide intra- and interindividual variability of cyclosporine pharmacokinetics and pharmacodynamics makes optimum immunosuppression difficult. Monitoring of blood cyclosporine concentrations has facilitated dosing individualization. Despite extensive clinical experience, the variability of cyclosporine pharmacokinetics and clinical response is not as yet thoroughly explained.

Cyclosporine, a cyclic polypeptide with a molecular weight of 1,203 daltons in addition to a high degree of lipophilicity and binding to lipoproteins, is absorbed predominantly in the small intestine. Oral cyclosporine was initially introduced in therapeutics as an oil-based formulation (Sandimmune^®). A high-fat meal offered concomitantly with a cyclosporine dose, which is dissolved in olive oil additionally diluted in milk or orange juice, is associated with a significant increase in cyclosporine area under the plasma concentration-time curve (AUC) compared with administration of a low-fat meal or vehicle alone in kidney transplant recipients. This enhancement is observed in both patients and healthy volunteers. A stimulation of bile flow, which accompanies dietary fat consumption, may be the contributing factor to the improved solubility and subsequent increased absorption of cyclosporine. This oil formulation is not ideal, as cyclosporine absorption is highly bile-dependent. Poor absorption through the gastrointestinal mucosa seems to be one of the main reasons for the low, variable bioavailability of cyclosporine.

The absorption of the conventional formulation of cyclosporine displays considerable inter- and intra-patient variability. Cyclosporine bioavailability varies between 20% and 60% and increases with time after kidney transplantation. This low, variable bioavailability renders it difficult to institute and monitor immunosuppressive therapy after organ transplantation. Better bioavailability of cyclosporine would facilitate immunosuppressive treatment.

Recently, a new galenic formulation of cyclosporine was introduced (Neoral^®), which is a water-free microemulsion of cyclosporine. The microemulsion creates micelles, which are absorbed in the small bowel without the presence of bile. This enhances the bioavailability of cyclosporine, especially after liver transplantation.

We observed an increase of adverse events with this new microemulsion formulation in Mexican patients who were receiving doses similar to those used with the conventional cyclosporine formulation. This higher incidence of adverse events may be due to a higher bioavailability of Neoral^® than that observed with the conventional formulations. In a previous study, our group reported that bioavailability of cyclosporine, administered as Sandimmune^®, was higher than the bioavailability reported in Caucasians. Therefore, it is important to compare the bioavailability of the two formulations of cyclosporine to design the adequate dosage regimens that should be used with the Neoral^® formulation in the Mexican population.

Twenty-three healthy volunteers (10 female and 13 male) participated in the study conducted according to the recommendations of the Helsinki Declaration and was approved by the local Ethics Committee of our Institution. All subjects gave written informed consent for participation. Volunteers were physically fit and no abnormalities were detected in routine clinical and laboratory tests. Hepatic, renal and cardiovascular disorders were excluded by medical history, physical examination, and suitable laboratory determinations. None of the subjects smoked, used drugs, abused alcohol, or was taking any medication.

The study was carried out according to a randomized crossover design, allowing a washout period of 1 week. After an overnight fast (10 h), subjects received the fat-rich diet customary in a Mexican breakfast (carbohydrates: 43.81%, proteins: 9.6%, and lipids: 45.71%) and were administered 7.5 mg/kg cyclosporine oral solution 30 min later (Group A, conventional solution, and Group B, Neoral^®, Sandoz de México, Mexico City) dissolved in 100 mL of orange juice. An indwelling cannula with a heparin lock was inserted into a forearm vein and 0.5-mL blood samples were drawn in tubes with EDTA anticoagulant at 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 h after medication. The fresh whole-blood samples were assayed for cyclosporine by specific monoclonal radioimmunoassay according to manufacturer instructions (Sandimmune Kit, Sandoz Ltd., Basel, Switzerland). This kit uses ³H-tracer and has a monoclonal antibody with high specificity for unchanged cyclosporine.

A compartment-independent pharmacokinetic analysis was used. Maximal whole blood concentration (C_max) and its time of occurrence (t_max) were compiled from the concentration-time data. AUC_0-24 was calculated by the linear trapezoidal rule.

To establish whether the formulations tested were bioequivalent, analysis of variance for a crossover design was carried out for logarithmically transformed bioavailability parameters (C_max and AUC_0-24). An additional evaluation of the ratio between test and reference formulations was calculated, as well as the 90%-confidence limits. The probability of obtaining values outside the limits of acceptance was determined by the two one-sided t test. Limits of acceptance for considering the two formulations tested bioequivalently were 80 to 125%.

It depicts the circulating concentrations against time curves obtained after administration of the two formulations assayed. It can be clearly seen that the microemulsion formulation was more rapidly absorbed, reaching C_max in 2.3 h, whereas the maximum observed with the conventional formulation was reached in 3.69 h. As a consequence of this faster absorption with the microemulsion formulation, a higher C_max was reached. However, extent of bioavailability, expressed as AUC_0-24, was about 10% higher with microemulsion than with the conventional formulation. Bioavailability parameters obtained with the two. In order to establish whether the two formulations are bioequivalent, the ratios (T/R, test/reference) for AUC and C_max obtained with the two formulations were calculated. Subsequently, 90%-confidence limits were estimated and the probability of obtaining values outside the limits of acceptance was calculated. It demonstrates the values obtained. It is clearly shown that the formulations are bioinequivalent, because the probability of obtaining values outside the limits of acceptance is higher than 0.05. In general, both treatments were well tolerated. No subject experienced clinically relevant changes in vital signs during the course of the investigation. Nausea and generalized burning sensations were the main adverse effects.

A comparison in the bioavailability of cyclosporine in two different oral formulations, microemulsion, and conventional solution, was carried out in healthy Mexican volunteers. In our study, a higher bioavailability with Neoral^® was observed, reflected in the increased values of both C_max and AUC as well as in a reduction of t_max. These results indicated that the microemulsion formulation is absorbed more rapidly than the conventional formulation.

Rocuronium is a non-depolarizing steroidal muscle relaxant with a short time to onset and of intermediate duration of action. The effects of an intravenous bolus dose of 600 ?g/kg of rocuronium in infants and children during halothane anesthesia have been previously described. Differences of response to neuromuscular blocking drugs occur in adults and children. Unfortunately, the time-course of rocuronium in children during isoflurane anesthesia has not been extensively evaluated. Because this anesthetic agent is commonly used in children, the time-course of the effect of rocuronium during isoflurane anesthesia needs to be further studied.

In addition, it has been suggested that age influences the neuromuscular response to muscle relaxants in children, but this has not been confirmed.

We performed the present study in order to evaluate the neuromuscular effect of 400, 600, and 800 ?g/kg of rocuronium administered to children during isoflurane anesthesia. We also assessed the relationship between the neuromuscular response and age (years) or weight (kg) of all children or children grouped by gender.

After approval by the Institutional Investigation Committee of the Hospital Infantil de México (Mexico City) and written informed parental consent, we studied 45 children aged 2–14 years, ASA physical status 1, undergoing elective surgery requiring tracheal intubation. No children were taking drugs known or suspected to interfere with neuromuscular transmission. No premedication was used in any patient. All children were randomly selected to receive either dose 400, 600, or 800 ?g/kg of rocuronium in a double-blind design.

On arrival at the operating room, children were monitored by means of ECG, pulse oxymetry and non-invasive blood pressure. Anesthesia was induced using atropine (10 ?g/kg), fentanyl (2 ?g/kg), and propofol (3 mg/kg). Normocapnia and normal body temperature (rectal or axillary) were maintained intra-operatively.

After induction of anesthesia, children were ventilated with 100% oxygen and neuromuscular function was monitored at the ulnar nerve using the TOF-guard neuromuscular transmission monitor (manufactured by Biometer International A/S, Odense, Denmark). The TOF-guard uses an accelometer (acceleration transducer) to measure the response to nerve stimulation and utilizes the Newtonian principle that acceleration is proportional to force. Using surgical tape, the acceleration transducer was attached to the flexor aspect of the freely mobile thumb and cutaneous electrodes to the ulnar nerve immediately proximal to the wrist joint. The TOF-guard was calibrated using its automatic start-up procedure and programmed to automatically deliver supramaximal train-of-four stimuli with square wave pulses of 0.2 msec duration to the ulnar nerve, in two phases. During the first, the stimuli was delivered every 15 sec from baseline to 5 min. Thereafter, the stimuli was delivered every 5 min from 5 min until complete recovery (T1:T0 = 100%) was observed. The response data were registered by one of the authors without knowledge of the dose of rocuronium.

The rocuronium (kindly donated by Organon-Teknika de México, Mexico City) was administered as a bolus dose into the T-connector of a rapidly running intravenous infusion. Simultaneously, the TOF-guard programmed sequence was initiated and the ratio of the height of the first response to the control height (T1:T0) was measured.

Finally, all children were intubated when either the T1:T0 reached 0% or there were no changes in the muscular relaxation present during a 1-min period. Endotracheal intubation condition was evaluated by another author also blinded for patient allocation group, using the four-score scale described by Fahey et al. Anesthesia was maintained after intubation with 1.5–2.0% isoflurane in 100% oxygen (flow rate of 4 L/min). Adequate anesthetic depth was judged by the lack of clinically significant changes in cardiovascular variables, i.e., heart rate and blood pressure, according to patient age. No additional drug was required during the anesthetic procedure.

For analysis, the response was also divided into two phases. The first was observed during the first 5 min and was used to compute the times to onset of action (TOA), while the second phase was from 5 min to complete recovery of neuromuscular function and was used to compute each time to spontaneous recovery of neuromuscular function (TSRNMF).

In order to obtain the TOA including time to 90 (B₉₀) and 99.9% (B₁₀₀) of relaxation or the TSRNMF including time to spontaneous recovery of 10 (T₁₀), 25 (T₂₅), 50 (T₅₀), 75 (T₇₅), and 90% (T₉₀) of neuromuscular function, T1:T0 response was fitted to time using SigmaPlot 4.01 (SPSS México, Mexico City) for Windows™ 95. The SigmaPlot curve fitter employs the Marquad-Levenberg algorithm to ascertain the parameters of the independent variable that provide the best fit between the equation and the data. The time-course of T1:T0 in each phase was considered to be sigmoidally related to time.

The model used was a four-parameter logistic equation where Y is the observed response and X, the time at which each response was registered. Additionally, a, b, c, and d are the four derived parameters where a = response at zero time, b = slope factor, c = time at which half maximal response is given, and d = maximal response.

The quality of fit of the pharmacodynamic (sigmoidal) model to the data was judged by the correlation coefficient automatically computed by SigmaPlot and by visual examination of both plots of observed vs. predicted response and plots of residuals (observed-predicted response) related to time.

All data were expressed as mean ± SD, median (ranges) and 95% confidence intervals for non-parametric data. The Fisher exact test was used for comparison of gender both among the three groups and in the general population. The comparisons of the TOA and the TSRNMF among the three doses were performed both parametrically by ANOVA and non-parametrically by the Kruskal-Wallis test followed by the Wilcoxon rank sum test to identify differences. Children were thereafter grouped by gender, and comparisons were performed by the Wilcoxon rank sum test to identify differences between males and females.

Linear regression analysis was performed between each TOA or TSRNF and the age or weight of all children. Thereafter, children were grouped by gender and linear regression analysis was performed between each TOA or TSRNMF and the age or weight previously significantly correlated while including all children. The 95% confidence intervals for the graphic linear relationship were automatically computed by SigmaPlot.

Multiple linear regression analysis was performed between each TOA or TSRNMF as the single quantitative dependent variable, and the age and weight of all children as quantitative explanatory variables. Finally, a two-tailed probability of p <0.05 was considered significant in each test or analysis.

In relation to the effect, the mean time-response courses of neuromuscular block and of spontaneous recovery of neuromuscular function observed with each dose of rocuronium. The TOA including B₉₀ and B₁₀₀ and TSRNMF including T₁₀, T₂₅, T₅₀, T₇₅, and T₉₀ from the three groups. The B₉₀ and B₁₀₀ were similar among groups, whereas all TSRNMF were similar between 600 and 800 ?g/kg of rocuronium, and significantly longer than those observed with 400 ?g/kg. In addition, all patients receiving 600 or 800 ?g/kg reached the maximal (100%) relaxation response, while three children in the 400 ?g/kg group reached 79, 96, and 98% inhibition, respectively, but were successfully intubated after 1 min of stabilization of muscular relaxation. The age and weight of the three children were 6, 2, and 8 years and 18, 9, and 21 kg, respectively. The three children were excluded for the comparisons of B₁₀₀, while only one was excluded for B₉₀ and T₁₀.

Great progress has been achieved in the study of pituitary tumors during the last two decades regarding early and adequate treatment; nevertheless, how to determine the effectiveness of treatment is still being discussed. In the particular case of acromegaly, the standard rule to assess efficacy of treatment is now the proportion of GH suppression following a glucose load; however, several investigators are exploring new tests that could be widely used as well as being less costly for assessing the somatotropic axis. It has been mentioned that overall GH output in acromegaly is often unrelated to either the duration of the disease or to the degree of symptoms. Some investigators have suggested that these parameters appear to correlate more closely with serum insulin-like growth factor–I (IGF-I). It is known that GH regulates the production of IGF-I in several tissues; in turn, IGF-I affects the pituitary secretion of GH. IGF-I is found in two major IGFBP complexes with molecular masses of 150 and 40 kD. Approximately 80% of the total IGF-I detected in sera is associated with the larger molecular complex and is completely saturated with IGFs. IGFBP-3 is the principal binding protein, sequestering about 75% of IGF-I and IGF-II in a large complex of 150 kDa. Both IGF-I and IGFBP-3 are GH-dependent, and therefore their measurement represents a stable index of long-term GH output. Because IGFBP-3 is GH-dependent and possesses a prolonged half-life but shows neither pulsatility nor circadian pattern, it could be an integrated marker of somatotroph function for the clinical evaluation of GH excess as well determining cure and recurrence in acromegaly. This study addresses the clinical usefulness of the measurements of both free and total IGF-I as well IGFBP-3 for the diagnosis of activity in acromegaly and the effectiveness of treatment in patients who have undergone transsphenoidal surgery.

The study included 13 patients aged 22–54 years with active acromegaly and who were treated by transsphenoidal pituitary surgery. The study was approved by the Ethics Committee of the Instituto Mexicano del Seguro Social, and all patients gave informed consent at the time of enrollment.. Baseline blood levels of GH, total IGF-I, free IGF-I, and IGFBP-3 were determined in all patients before a 75-g glucose load test was carried out prior to surgery and 3 months after transsphenoidal surgery. Glucose suppression was considered abnormal if GH failed to suppress to 2 ?g/L or less at any time points (30, 60, 90, or 120 min) after the administration of glucose load. The presence of somatotroph adenoma by immunocytochemistry analysis was confirmed in all cases. In addition, 6 of the 13 cases had positive cell immunochemistry for prolactin. The control group was comprised of 14 normal subjects who were age-, weight-, and sex-matched.

Blood samples were collected in a 10-mL vacuum tube between 7 and 8 h after an overnight fast and were centrifuged at 2,500 rpm for 10 min. Serum aliquots were separated, and all samples were maintained frozen at ?35°C until assayed in a single-assay run for each biochemical marker. GH was measured by a polyclonal double antibody RIA using a commercial kit (Diagnostic Products Co., Los Angeles, CA, USA). The sensitivity for this assay was 0.7 ng/mL, and the intra- and interassay coefficients of variation (CV) were 2.7 and 4.2%, respectively, as previously reported. Total IGF-I, free IGF-I, and IGFBP-3 were determined by two-site immunoradiometric assay (IRMA) using commercial kits (Diagnostic System Laboratories, Webster, TX, USA). Measurement of total IGF-I included an acid-ethanol extraction step to separate IGF-I from its binding protein. The sensitivity for this assay was 0.80 ng/mL, and the intra- and interassay CV were 2.6 and 4.4%, respectively. Free IGF-I had a sensitivity of 0.03 ng/mL, and the CV were 6.2 and 7.3%, respectively; as to IGFBP-3, the sensitivity was 0.5 ng/mL, and the CV were 1.9 and 3.9%.

All data are given as medians and ranges. The significance of any difference was analyzed using Wilcoxon-Whitney’s test. Spearman’s correlation analysis between different biochemical markers was performed.

Before patients were treated by transsphenoidal pituitary surgery, baseline levels of GH, of free and total IGF-I, as well as of IGFBP-3 were consistently elevated; all biochemical values significantly decreased after surgery. It was also observed that glucose load test before surgery was unable to decrease serum GH levels; likewise, free IGF-I and IGFBP-3 levels remained elevated. It shows the correlation analysis in controls, acromegaly, and in patients who exhibited either normal or abnormal suppression of GH levels with the glucose load test performed after pituitary surgery. In addition, IGFBP-3 demonstrated to be an accurate index because of its close correlation with GH suppression by glucose load test (0.91, p <0.001). In seven out of a total patients, the provocative glucose test resulted in a fall of GH to levels of less than 0.7 ng/mL three months after surgery; four of them had normal plasma IGFBP-3 and total IGF-I levels, and only three showed normal plasma IGF-I levels. Only IGFBP-3 after surgery was significantly higher in those patients who did not suppress their GH levels to <0.7 ng/mL compared to those who did. All patients exhibited a high correlation between IGFBP-3 levels and GH suppression induced by glucose. A negative correlation (r = ?0.67, p <0.01) between IGFBP-3 and free IGF-I was observed in all 13 patients at the time of enrollment before treatment; however, this correlation became positive (r = 0.54) 3 months after surgery. Postoperative total and free IGF-I levels were similar in those patients who suppressed their GH to levels of less than 0.7 ng/mL and those who did not. However, IGFBP-3 levels were consistently augmented in acromegalic patients before treatment, and uniformly decreased in treated patients who simultaneously exhibited GH levels below 0.7 ng/mL after glucose load. No correlation between total and free IGF-I was observed before treatment, but this correlation (r = 0.04) became significant (r = 0.60, p <0.05) after surgery.

Results of the present studies have shown that baseline serum levels of IGFBP-3 are consistently elevated in patients with active acromegaly and that they did not overlap with values observed in normal controls. Both IGF-I and IGFBP-3 are GH-dependent peptides that are elevated in patients with active acromegaly; both reflect the integrated secretion of GH. Our study showed that IGFBP-3 values correlate closely with the degree of GH suppression by glucose load test; conversely, baseline serum levels of total and free IGF-I did not show the same correlation with the amount of GH suppression obtained with the glucose load. From these results, it might be suggested that a single IGFBP-3 blood determination is a reliable biochemical marker in the initial study of patients with acromegaly. Additional studies are necessary to establish the precise cut-off limit for the IGFBP-3 value to obtain the diagnostic reference. After pituitary surgery, IGFBP-3 estimations showed values similar to those obtained by GH suppression by the glucose load test to establish acromegaly activity and effectiveness of surgical removal of the tumor. However, the criteria for GH suppression has changed considerably over the last decade; with the recent development of ultrasensitive GH assays, it has been shown that upon a glucose load, normal individuals suppress their GH levels to 0.029 ng/mL in males, and to 0.25 ng/mL in females. Because the glucose load test requires repeated blood samples for GH determinations as well as close vigilance of the patient, it may be advantageous to obtain a single basal determination of IGFBP-3 and IGF-I for diagnosis of acromegaly and to determine the effectiveness of transsphenoidal pituitary surgery.

It is well known that activated macrophages generate a variety of cytotoxic factors, oxygen radicals, and nitric oxide. Nitric oxide is considered the major effector against tumoral cells. Even though activated macrophages are efficient tumoral cell killers, this macrophage property is abridged in some patients and experimental animals bearing malignant tumors. We previously observed that in L5178Y tumor-bearing mice, the macrophages converging at the ascites tumor did not exhibit tumoricidal activity against malignant lymphoblasts. However, after the tumoral cells were killed or severely injured (as a result of a chemotherapeutic experimental schema), the associated macrophages from the ascites tumor became activated against L5178Y lymphoblasts. In addition, Wiltrout et al. showed that tumor-associated macrophages recovered from L5178Y lymphoma-bearing mice conserve their entire capacity and could be activated with LPS-killed malignant lymphoblasts in vitro. The previously mentioned actions strongly suggest that intact tumoral cells release certain molecules that reversibly inhibit macrophages from tumor-bearing animals. Accordingly, the objectives of the present study were the following: (a) to determine whether the activation of macrophages with LPS, IFN-?, or a combination of both were prevented by mouse cell-free ascitic liquid (CFAL) or the soluble fraction (S1) from L5178Y lymphoblast lysates, and (b) to investigate whether macrophages from healthy or tumor-bearing mice were susceptible to inhibition by CFAL or S1.

The L5178Y murine cell line was used in this study. This is a transplantable murine leukemia cell line, which was derived in 1952 by LW Law from a thymic tumor induced in a DBA/2 mouse by methylcholanthrene. This cell line has been maintained for the last 20 years in our laboratory by serially inoculating the lymphoblasts into BALC/c mice (see subsequent text).

Two-month-old male BALB/c mice were used to both maintain the tumoral cell line and as a source of normal- and tumor-bearing peritoneal macrophages. The animals were kept in polypropylene cages with food (Lab Diet 5001, PMI Nutrition International, Inc., Brentwood, MO, USA) and water ad libitum under controlled conditions of light, humidity, and temperature.

The mice were serially inoculated as follows: one healthy male BALB/c mouse, 2 months of age, was inoculated intraperitoneally with 100 ?L of ascites liquid containing 2 × 10⁷ lymphoblasts. Twelve days later, the ascitic liquid was harvested and used to inoculate a new mouse, and so on.

Four mice were injected i.p. with 2 mL of 4% thioglycollate (Merck de México, Mexico City, Mexico) in phosphate-buffered saline; (K₂HPO₄ 0.02 M, KH₂PO₄ 0.02 M, NaCl 0.12 M, pH 7.4; PBS). Ninety-six hours later, the macrophages were harvested by peritoneal cavity lavage with RPMI-1640 medium (Sigma Chemical Co., St. Louis, MO, USA). The liquid obtained was centrifuged at 450 × g for 10 min at room temperature. The supernatant was discarded and the cellular pellet resuspended in 4 mL of RPMI-1640 fresh medium. Each of the previously mentioned materials was placed on a 4-mL NycoPrep^™ 1.068 layer (Nycomed Pharma Centrifugation Media, Oslo, Norway) and the preparation was centrifuged at 600 × g for 10 min at room temperature. The macrophages were recovered from the ascites lymphoma liquid with NycoPrep^™ 1.068 as recommended by the manufacturer. In brief, 4-mL ascites lymphoma liquid was placed in a 13 × 100 mm glass assay tube containing a 4-mL NycoPrep^™ 1.068 layer. This preparation was centrifuged at 600 × g for 10 min at room temperature. Thereafter, three bands were formed in the tube. The central tube, placed just above the pellet, contained the macrophages. To recover the macrophages, the upper layer (3–4 mm wide) was extracted with a vacuum and discarded. The layer below was carefully extracted with a Pasteur pipette, placed into a clean assay tube, and two volumes of fresh RPMI-1640 medium were added and centrifuged as before. The supernatant was discarded and the macrophage pellet was resuspended and washed twice with RPMI-1640 medium. A sample from these cells was dyed with Wright’s stain, and the cells’ major monocyte/macrophage characteristics were verified as follows: reniform or fusiform cells sized 15–20 ?m with a dark violet-colored nucleus and one or two nucleoli, showing light-blue vacuolated cytoplasm with multiple, fine pink-purple granules. This preparation density was brought to 1 × 10⁶ cells/mL with RPMI-1640 medium plus 10% fetal bovine sera. Afterward, each of the 96 wells from a microplate (Sigma Chemical Co.) was inoculated with 200 ?L of the previously mentioned cell suspension and the microplate was incubated at 37°C in a 5%-CO₂ atmosphere for 24 h.

Two milliliters of ascitic liquid was harvested by aspiration from a 12-day-old tumor-bearing mouse. The liquid was placed in a 13 × 100 mm glass assay tube containing 4 mL of NycoPrep^™ 1.068 layer. The macrophage band was saved and the cells washed twice with RPMI-1640 medium. Monocyte/macrophage lineage was confirmed by Wright staining, as carried out with normal macrophages. Cell number was determined with a hemocytometer, and their density adjusted to 1 × 10⁶ cells/mL with RPMI-1640 medium plus 10% fetal bovine serum. Subsequently, 200 ?L of the lymphoma cell suspension were poured into each of the 96 wells of a microplate and incubated at 37°C in 5%-CO₂ atmosphere for 24 h.

The ascitic liquid from two mice bearing a 12-day lymphoma was pooled (4 mL) and centrifuged at 600 × g for 10 min at room temperature. The supernatant (CFAL) was saved and stored at ?20°C until used. The cell pellet was mixed with a 4-mL lysis solution (1 mM EDTA, 1 mM epsilon amino caproic acid, and 1 mM dithiothreitol [Calbiochem, San Diego, CA, USA]). This preparation was frozen at ?20°C for 24 h and thawed at room temperature. The suspension was shaken vigorously with a vortex and centrifuged at 600 × g/10 min. The supernatant (S1) was stored at ?20°C until used.

The microplates containing adherent macrophages were incubated in the presence of 5 ?g/mL lipopolysaccharides (LPS), 40 U/mL IFN-? (Sigma Chemical Co.), or a LPS (5 ?g/mL)/ IFN-(? 40 U/mL) blend. The quantity of nitric oxide released by the macrophage cultures was quantified in their cell-free supernatant after 48 h of incubation in the presence or absence of tumor fractions according to the Griess method12. D.J. Stuehr and C.F. Nathan, Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 169 (1989), p. 1543. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (867). Optical density measurements at 545 nm were carried out with a microplate reader (ElA Multi-Well Reader, Sigma Diagnostic (St. Louis, MO, USA). All determinations were done in triplicate.

MDA was assayed as follows: the macrophage cultures (as described in Materials and Methods), were supplemented with 5 ?g LPS/mL, 40 U IFN-?/mL, or a combination of 5 ?g LPS U/mL and 40 IFN/mL; these preparations were incubated at 37°C for 24 h; 10 ?L of CFAL or S1 were added; the plates were reincubated for 24 h, and the amount of nitric oxide determined in the spent culture medium.

MAIA assays were carried out as follows: the macrophage cultures were added with 10 ?L of CFAL or tumor fractions and incubated at 37°C; after 24 h at 5 ?g LPS/mL, 40 U IFN-?/mL, or a combination of 5 ?g LPS U/mL and 40 IFN/mL were added and reincubated at 37°C for 48 additional hours, and the amount of nitric oxide released by macrophages was determined in their spent culture medium.

Progesterone exerts multiple actions on the nervous system, including a neuroprotective action against cerebral infarct caused by experimental occlusion of the middle cerebral artery in rats, the promotion of myelination in the central and peripheral nervous systems, and the possession of anticonvulsant properties through potentiation of the GABA-evoked Cl^? currents.

From its striking behavioral actions, the anxiolytic properties of progesterone have long been recognized in humans. Similarly, the exogenous administration of progesterone in estradiol-primed rats reduces anxiety8. J.F. Rodríguez-Sierra, J.L. Howard, G.T. Pollard and S.E. Hendriks, Effect of ovarian hormones on conflict behavior. Psychoneuroendocrinology 9 (1984), p. 293. Abstract | View Record in Scopus | Cited By in Scopus (42). Low levels of anxiety are concurrent with high levels of circulating progesterone occurring spontaneously throughout the estrous cycle and pregnancy. In addition, progesterone produces some anxiolytic actions in both estrogen- and non-estrogen-primed rats. However, while some antidepressant drugs produce anxiolytic effects, some anxiolytics also produce antidepressant actions. Progesterone consistently restores immobility to control values in the tail suspension test in ovariectomized mice; however, the action of progesterone in other tests that measure the antidepressant activity of drugs more specifically remains to be explored.

In the present study, we describe the changes that exogenous administration of progesterone elicited in the immobility measured in rats forced to swim. The validity of this experimental model in monitoring the potency of antidepressant drugs has been extensively discussed, the conclusion being that antidepressants reduce immobility, with few or no effects on locomotor activity. This is a different action from that exerted by anxiolytics, because these compounds commonly reduce locomotor activity and increase immobility in the forced swim test. We, therefore, tested the effect of ovariectomy and the administration of progesterone in rats forced to swim.

Wistar rats (aged 3 months, weighing 250–300 g) were used for this study following strict principles of animal care (NIH publication No. 86-23, revised 1985). The rats were assigned to one of two groups, vehicle or progesterone, and housed 4 to 5 rats per cage at a constant room temperature (25 ± 1°C) under a 12-h light-dark cycle (lights on at 7:00 h) with water and food freely available. All animals were anesthetized with ethylic ether and ovariectomized (OVX) from a ventral approach. After surgery, the animals were placed in their home cages for recovery and checked once daily to avoid any additional discomfort.

In both groups of rats, a 15-min pretest habituation session for locomotor activity and forced swim preceded the experimental sessions by 24 h. The first control test (C1) was applied 1 week before ovariectomy, with the majority of the rats in diestrus (80%); the second test was done 2 weeks after surgery (OVX), and from the third through the seventh tests, the animals received saline (NaCl 0.9%) and water-soluble progesterone (Sigma Chemical Co., St. Louis, MO, USA). Finally, the eighth test was done 1 week after the last injection of progesterone or saline (C2). In the experimental group (n = 9), each rat underwent once-weekly i.p. injections of water-soluble progesterone in a volume of 0.10 mL (NaCl 0.9%). Progesterone (0.20, 0.40, 0.80, 1.50 and 3.0 mg/kg) was injected 24 and 2 h before behavioral testing (between 10:00 and 12:00 h). To discard the cumulative effects of progesterone and a possible effect from the swimming test repetition, all animals received one of several progesterone doses in a different sequence, following a completely randomized experimental intrasubject design. The control group (n = underwent 8 once-weekly swim test sessions. In order to discard a possible influence of progesterone on locomotor activity impinging on the forced swim test, the open field test preceded the measurement of immobility in the forced swim test. The apparatus consisted of an opaque-Plexiglas box (40 × 30 × 20 cm). A black grid divided the floor into 12 equal squares (10 × 10 cm). The animals were placed in a corner of the apparatus and the number of times an animal entered a square on four paws during a 5-min videotaped session was counted by two independent observers (any discrepancy implied a re-analysis of the videotape).

Immediately thereafter, the forced swim test was practiced individually in a glass cage (50 × 30 × 60 cm) containing water (25 ± 1°C) at a depth of 19–21 cm, depending on the head-tail length of each rat. After the swimming session, the animals were allowed to recover in a warm, dry chamber. The 5-min videotaped test sessions took place the next day until eight weekly experimental sessions were completed. A rat was judged immobile when it remained floating in the water while making only the necessary movements to keep its head above water. Two observers unaware of the treatment evaluated the total time of immobility.

Because the data failed to follow a normal distribution, the results were analyzed from dosage and treatments by the Friedman test. The Student-Newman-Keuls test was used when at least p <0.05 was attained. The results are expressed as mean ± standard error of the mean.

In the open-field test, ovariectomy lowered by 50% the crossing amount observed in the session practiced before the surgery (p <0.05), but progesterone failed to produce changes in crossing after ovariectomy even at the highest dose tested (3.0 mg/kg).

Ovariectomy (OVX) did not produce any significant change in immobility in the forced swim test. Similarly, the weekly repetition of the test produced non-significant changes consisting of some trends to decrease or increase immobility in the saline-treated group throughout the 8 weeks of study, but in any case these oscillations reached the criterion of significance.

In the forced swim test, the total mean duration of immobility throughout the study (eight sessions) significantly decreased (p <0.05) in the progesterone group (16.4 ± 1.85 sec) with respect to the saline group (25.3 ± 5.55 sec), but in the analysis of the weekly sessions, a trend toward decreased immobility at a dose of 0.40 mg/kg of progesterone was found. A dose of 0.80 mg/kg significantly decreased immobility (p <0.001) to less than 30% of that obtained in the control test practiced after ovariectomy. In the post-treatment test practiced 1 week after the last injection of progesterone, the immobility values (C2, 25.3 ± 9.44) returned to the values observed in the control test (C1 23.1 ± 3.88; OVX 27.0 ± 6.23) recorded prior to surgery.

Total time in immobility. Progesterone reduced immobility from a dose of 0.80 mg/kg and immobility returned to control values (C2) 1 week after the last injection of progesterone. Open symbols correspond to saline group and filled symbols to progesterone group. ^• p <0.05 vs. C1, OVX and control group.

In this study, we explored some of the antidepressant-like action of progesterone by using the forced swim test. Progesterone shortened the total time spent during immobility in the forced swim test, but failed to show any significant action on locomotor activity, thus behaving as an antidepressant at the dose tested.