Polymerase chain reaction (PCR) detection of B cell clonality in Sjögren's syndrome patients: a diagnostic tool of clonal expansion.

Sjögren's syndrome (SS) is an autoimmune disease characterized by clonal B cell attack of the exocrine glands and dysregulated expression of B cell-activating factor (BAFF). Based upon the current data of increased rates of lymphoid malignancy, as non-Hodgkin's lymphoma (NHL) is associated with SS, we propose the detection of clonal rearrangements of immunoglobulin heavy chain (IgH) gene in those patients as a predictor of malignant clonal expansion. To test our proposal, we examined the IgH clonal rearrangements in SS patients (60) and healthy control subjects (42) having chronic non-specific sialadenitis, to determine the presence of clonal B cells in minor labial salivary glands (MSG) of SS patients. Clonal B cell expansion was assessed by two polymerase chain reaction (PCR) assays: (i) semi-nested PCR, against sequences encoding framework regions FR3, FR2 and FR1c of the variable chain IgH gene in B cells present in the MSG infiltrate; and (ii) the PCR-enzyme-linked immunosorbent assay (ELISA) technique, against the major and minor breakpoint regions of the Bcl-2 oncogene coupled with a variable segment of the IgH to assess the Bcl-2/JH translocation. When FR3, FR2 and FR1c primers were employed, we detected B cell monoclonality in 87% of the SS patients and 19% of the control subjects. The association between inflammation severity of the MSG pattern and the presence of B cell clonality was found to be statistically significant (P<0.01). We concluded that the presence of B cell clonality in MSG can be used as a index of an altered microenvironment favouring the development of lymphoma in SS patients.

Proximal amputation of inferior extremity secondary to recurrent pressure ulcers in patients with spinal cord injuries.

STUDY DESIGN: Descriptive case series study. OBJECTIVE: To describe the course of five spinal cord injury (SCI) patients who underwent proximal amputation of the inferior extremity, secondary to recurrent, complicated pressure ulcers (PU) and the clinical impact this intervention had in these patients. PLACE: Trabajador Hospital in Santiago, Chile. METHOD: Revision of five clinical cases of patients who underwent partial hemipelvectomy or hip disarticulation with amputation of the extremity as treatment for pelvic recurrent PU with chronic secondary osteomyelitis. The clinical impact was quantified as days of hospital stay, number of surgeries and previous and post surgery PU. RESULTS: After the proximal amputation of the extremity, patients significantly decreased number of days of hospital stay (P=0.035), number of surgeries (P=0.015) and PU (P=0.0065). CONCLUSION: Partial hemipelvectomy and hip disarticulation with proximal amputation of the inferior extremity are rescue procedures that can be last resource treatment for chronic recurrent pelvic PU secondary to chronic osteomyelitis.

Functional activity of eukaryotic signal sequences in Escherichia coli: the ovalbumin family of serine protease inhibitors.

It is widely assumed that the functional activity of signal sequences has been conserved throughout evolution, at least between Gram-negative bacteria and eukaryotes. The ovalbumin family of serine protease inhibitors (serpins) provides a unique tool to test this assumption, since individual members can be secreted (ovalbumin), cytosolic (leukocyte elastase inhibitor, LEI), or targeted to both compartments (plasminogen activator inhibitor 2, PAI-2). The facultative secretion of PAI-2 is mediated by a signal sequence proposed to be inefficient by design. We show here that the same internal domain that promotes an inefficient translocation of murine PAI-2 in mammalian cells is a weak signal sequence in Escherichia coli. In contrast, the ovalbumin signal sequence is much more efficient, whereas the corresponding sequence elements from LEI, maspin and PI-10 are entirely devoid of signal sequence activity in E.coli. Mutations that improve the activity of the PAI-2 signal sequence and that convert the N-terminal regions of maspin and PI-10 into efficient signal sequences have been characterized. Taken together, these results indicate that several structural features contribute to the weak activity of the PAI-2 signal sequence and provide new insights into the plasticity of the "hydrophobic core" of signal sequences. High-level expression of two chimeric proteins containing the PAI-2 signal sequence is toxic, and the reduced viability is accompanied by a rapid decrease in the membrane proton motive force, in ATP levels and in translation. In unc- cells, which lack the F0F1 ATP-synthase, the chimeric proteins retain their toxicity and their expression only affected the proton motive force. Thus, the properties of these toxic signal sequences offer a new tool to dissect the interactions of signal sequences with the protein export machinery.

[Prevalence of sexually transmitted diseases and the use of the condom among sex workers]. / Prevalencia de enfermedades de transmisión sexual y uso del condón en sexoservidoras.

OBJECTIVE: To determine the prevalence of sexually transmitted diseases (STD) and the use of condoms by sex workers, assisted in a health module. MATERIAL AND METHODS: A cross sectional study, was performed with prostitutes in Coatzacoalcos, Veracruz through a self-administered questionnaire in a period from January 3 to February 9, 2001. The questionnaire evaluated gynecology-obstetric background, STD and the use of condoms. Clinical records and laboratory tests were evaluated for STD screening. RESULTS: A total number of 196 sex workers from 71 work places were evaluated. The average age was 29.5 +/- 3.3 years. One hundred and eleven (57%) reported being single. 189 (96.4%) presented at least a case of vaginosis during the year 2000 and 148 (75.5%) were diagnosed having vulvovaginitis due to fungus. Syphilis was identified in 4 (2%) cases and (0.5%) acquired immune deficiency syndrome. 45 (23%) reported they always use a condom and 113 (58%) use condoms very often. CONCLUSIONS: There is a high prevalence of vulva and vagina disorders, as well as syphilis. High rates of sex workers use condoms.

Management of the dental patient with pulmonary tuberculosis.

Tuberculosis is a potentially fatal infectious disease. Although it typically manifests in populations subjected to crowding and living in poor conditions of hygiene, in the past decade the morbidity and mortality associated to pulmonary tuberculosis has increased throughout the world--particularly due to the appearance of strains resistant to a range of drugs, and to the vulnerability of human immunodeficiency virus (HIV)-positive individuals to tuberculosis infection. In this context, dental professionals should implement schemes and protocols designed to identify patients with lung tuberculosis, in order to both refer such patients to specialized medical care and to avoid infection of the dental personnel and other patients. A review is made of the etiology, pathogenesis, clinical manifestations, diagnosis and treatment of patients with pulmonary tuberculosis, with particular emphasis on the management measures indicated in the dental clinic.

[Prevalence of breast self-examination in health workers. Factors associated with its correct performance]. / Prevalencia del autoexamen mamario en trabajadoras de la salud. Factores asociados a su correcta realización.

OBJECTIVE: Determine the frequency of a breast self-examination (BSE), in health workers and the associated risk factors in its correct performance. MATERIAL AND METHODS: From January 24 to February 24, 2000, a transversal analytical study was performed in the workers of the General Hospital Zone number 32, of the IMSS in Minatitlán, Veracruz. 106 women were included, who verbally accepted to participate in the study. A self-administered survey was applied which contained variables of the use and performance of the BSE, social demographic variables, information about BSE, and positive health attitudes and faces with breast cancer. RESULTS: A total of 92 (86.8%) of the workers have performed an BSE at least once in their life; 46.2% performed it adequately and 53.8% did not perform the technique adequately. The variables: profession, high social economical level, nulliparous after 25 years old, users of contraceptives, having visited a gynecologist for breast clinical examination, and if a family member recommended to perform a BSE, were related with the use adequately of BSE (p < 0.05). CONCLUSIONS: The frequency of the use of a breast self-examination is high, however, it is not performed adequately by the majority of the women studied. Its correct execution is associated with the following variables: medical background doctor or nurse, well informed about BSE, family support and a positive health attitude. It is necessary to perform educational interventions so that the breast self-examination can be performed correctly.

Genetic footprinting in bacteria.

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.

Estimulación magnética transcraneal y trauma acústico / Transcranial magnetic stimulation and acoustic trauma

La estimulación magnética transcortical (TCMS) es un método no invasivo para el estudio de la función motora en humanos, sin embargo, existen algunos autores que sugieren que causa daño auditivo (coclear). Se estudiaron 15 pacientes (edades entre 11 meses y 16 años, con una media de 6.8 años) con potenciales evocados auditivos de tallo cerebral (PEATC), emisiones otoacústicas (EOAs), reflejo estapedial (RE) y audiometría convencional cuando fue posible, antes y después de TCMS para estudios de conducción central en enfermedades neurológicas diferentes. Los pacientes no tuvieron protección auditiva ni historia de crisis convulsivas. Los potenciales evocados motores fueron registrados de los músculos abductores breves y primer interóseo dorsal en reposo y durante la contracción voluntaria cuando esto fue posible. Un promedio de 18 estímulos fue aplicado con intensidades entre 50 y 75 por ciento (11.5 Teslas de intensidad). Los PEATC, EOAs, RE y audiometrías fueron practicados antes y después de la estimulación, dos semanas después y dos meses después cuando fue posible. La distribución logarítmica natural tuvo disposición normal. No existieron diferencias significativas en las pruebas de función auditiva. Por tanto, es un procedimiento seguro para la integridad de la vía auditiva.

[Effectiveness and safety of mebendazole compared to nitazoxanide in the treatment of Giardia lamblia in children]. / Eficacia y seguridad de mebendazol contra nitazoxanida en el tratamiento de Giardia lamblia en niños.

OBJECTIVE: Compare the effectiveness and safety of mebendazole versus nitazoxanide in the treatment of Giardia lamblia in children. Giardiasis is an intestinal protozoan of worldwide distribution which most frequently affects the infantile population. In Mexico we have, found a frequency of three to sixty percent. We have, used different medications in it's treatment, but the experience with mebendazole and nitazoxanide is scarce. METHOD: An experimental study as a clinical assay. We included children from the ages of 4 to 12 years old, which had a positive Giardia lamblia cysts in their feces. The children were divided into to two groups: A, were a administered 100 mg of mebendazole every 12 hours, for three days; B, were administered 100 mg of nitazoxanide every 12 hours, for three days; A feces control study was performed at three, five and seven days post treatment. At the end of the treatment we asked the parents if the children had presented any adverse events during the administration of the medication. For the statistical analysis we used Student's t and Chi squared. RESULTS: We studied 82 children, 41 (50%) for each group. In group A, the control feces studies were negative 33 resulting in a 80.4% effectiveness; in group B, 32 were negative resulting in a 78.0% effectiveness, without being statistically significant with a p = 0.8. We found adverse, events in 9 (22%) of the children in group A and 16 (39%) in group B, there was, statistically significant difference with p = 0.09. However, we discovered that the children who received nitazoxanide suffered from abdominal pain more frequently. CONCLUSIONS: We can conclude that both mebendazole and nitazoxanide are efficient for use against infection due to Giardia lamblia, however, the secondary reactions with nitazoxanide were more frequent than with mebendazol.

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region.

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro.

Localization of the Escherichia coli cell division protein Ftsl (PBP3) to the division site and cell pole.

FtsI, also known as penicillin-binding protein 3, is a transpeptidase required for the synthesis of peptidoglycan in the division septum of the bacterium, Escherichia coli. FtsI has been estimated to be present at about 100 molecules per cell, well below the detection limit of immunoelectron microscopy. Here, we confirm the low abundance of FtsI and use immunofluorescence microscopy, a highly sensitive technique, to show that FtsI is localized to the division site during the later stages of cell growth. FtsI was also sometimes observed at the cell pole; polar localization was not anticipated and its significance is not known. We conclude (i) that immunofluorescence microscopy can be used to localize proteins whose abundance is as low as approximately 100 molecules per cell; and (ii) that spatial and temporal regulation of FtsI activity in septum formation is achieved, at least in part, by timed localization of the protein to the division site.

Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli.

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.

The mobilization protein, MobM, of the streptococcal plasmid pMV158 specifically cleaves supercoiled DNA at the plasmid oriT.

The streptococcal plasmid pMV158 replicates by the rolling circle mechanism. It encodes a relaxase protein of 494 residues, termed MobM, involved in conjugative mobilization. MobM protein was overproduced, purified, and shown specifically to relax supercoiled pMV158 DNA. The 5'-end and the 3'-end of the nick site introduced by MobM have been determined by sequencing and by primer extension analysis. The nucleophilic attack exerted by MobM is in the 5'-GpT-3' dinucleotide, within the sequence 5'-TAGTGTG/TTA-3'. Upon cleavage, MobM protein remains tightly associated with its target DNA, probably through a covalent bond. The pMV158 oriT did not exhibit homologies with known origins of transfer of plasmids from Gram-negative bacteria. However, several plasmids from Gram-positive hosts have a region identical or very similar to the pMV158 oriT. To our knowledge, this is the first demonstration of a relaxase activity of a mobilization protein from a plasmid replicating by the rolling circle mechanism.

A comparative, randomized trial of UFT and 5-fluorouracil in combination with cyclophosphamide and doxorubicin in the treatment of advanced breast cancer patients at The Philippines General Hospital.

A comparative, randomized trial was conducted to determine the efficacy of oral UFT (Tegafur and Uracil) versus 5-fluorouracil (5-FU) in combination with cyclophosphamide and doxorubicin in patients with metastatic breast cancer. Of 62 evaluable patients, 31 received UFT (350 mg/m2/day orally x 14 days), doxorubicin (50 mg/m2 intravenously [I.V.] day 1) and cyclophosphamide (500 mg/m2 I.V. day 1). The other 31 patients received 5-FU (500 mg/m2 I.V. days 1 and 8), doxorubicin (50 mg/m2 I.V. day 1), and cyclophosphamide (500 mg/m2 I.V. day 1). Regimens were repeated for a total of six cycles. The two groups were comparable in terms of age, gender, performance status, menopausal status, and number and sites of metastases. No statistical difference in overall response rates was seen (UFT arm, 48.4% vs. 5-FU arm, 35 %; p = 0.30). Median response duration was 16 weeks (range, 4-30) for both arms. The toxicity profile (alopecia, anemia, leukopenia, thrombocytopenia, diarrhea) was similar in both groups and both regimens were well tolerated. Anemia and stomatitis were significantly more common in the 5-FU arm (p = 0.02). Thus, oral UFT has response rates and duration of response that are comparable to 5-FU in a combination regimen for advanced breast cancer.

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.

We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

Changes in viral expression and cytokine profile induced by a polyantigenic immunomodulator in HIV-infected peripheral blood mononuclear cells.

This is the first time, to our knowledge, that evidence is presented showing that a polyantigenic immunomodulator (PAI), acting as a biological response modifier, can either induce or suppress HIV expression depending on the viral load of infected PBMC. PAI consists of a mixture of inactivated bacteria with influenza virus vaccine. PBMC from HIV-infected patients (asymptomatic, age 22-36, symptomatic, age 30-59 and pediatric, < 2 years old) were co-cultured with PHA-stimulated PBMC from uninfected individuals in medium containing IL-2 and PAI. Parallel co-cultures were carried out in a PAI-free medium. Cultures were fed with PHA-stimulated PBMC from uninfected donors on a weekly basis. HIV-p24 ag and cytokine profiles (IL-1 beta, IL-2, IL-4, IFN-gamma and TNF-alpha) were determined on supernatants on day 14. Peripheral blood samples from each patient were evaluated at the beginning of the experiment as to total CD3, total CD19, CD3/CD4, CD3/CD8, CD16/CD56, CD8/HLA-DR and CD8/CD38 markers through flow cytometry. PAI was able to induce viral expression (up to 11,881 pg/ml of p24 antigen) in cultures showing a low (less than 16 pg/ml) or no viral titer. In contrast, in those cultures with high viral titer (10(2)-10(5) pg/ml), a substantial reduction on the titer was observed upon exposure to PAI. PAI was able to induce the production of IFN-gamma and TNF-alpha while that of IL-4 and IL-1 beta was reduced. The predominant cell type detected in the blood samples of the studied subjects were CD8+, CD8+/CD38+ or CD8+/HLA-DR+.(ABSTRACT TRUNCATED AT 250 WORDS)