Enhancing the immunotherapeutic potential of mycobacteria by transfection with tumour necrosis factor-alpha

In an attempt to enhance the anti-tumour properties of mycobacteria we have developed recombinant forms of Mycobacterium smegmatis which express and secrete biologically active human tumour necrosis factor-alpha (TNF-alpha). This was achieved by transfecting M. smegmatis using shuttle plasmids incorporating the cDNA sequence for the human TNF-alpha mature peptide. In vitro experiments on a panel of human bladder tumour cell lines (EJ18, MGH-U1, RT4, RT112) indicate that our genetically modified mycobacteria are more effective than wild-type at inducing or up-regulating the expression of intracellular adhesion molecule-1 and the secretion of an array of proinflammatory cytokines [interleukin-1 (IL-1), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor]. We have also demonstrated increased adhesion molecule and cytokine expression in response to mycobacteria transfected with vector containing no gene insert. However, this was not as pronounced as that observed following tumour cell stimulation by the TNF-alpha-transfected strain. In contrast, in three out of four tumour cell lines all M. smegmatis strains were found to down-regulate the secretion of the anti-inflammatory cytokine transforming growth factor-beta1. Our studies have also confirmed that M. smegmatis is a powerful inhibitor of bladder tumour cell growth and revealed that its antiproliferative potency is enhanced by transfecting with human TNF-alpha and, to a lesser extent, with vector alone. All M. smegmatis strains were effective in the activation of peripheral blood leucocyte cultures. However, no differences were observed in the ability of the TNF-alpha-transfected, mock-transfected and wild-type mycobacteria to induce tumour cell killing activity. These results suggest that the immunomodulatory effects of M. smegmatis can be enhanced by transfection with vectors which allow the secretion of human TNF-alpha, thus increasing mycobacterial immunotherapeutic potential.     

Prevalence in a volunteer population of pelvic cancer detected with transvaginal ultrasound and color flow Doppler.

Our objective was to find the prevalence of non-symptomatic endometrial and ovarian neoplasms in a volunteer population of women, aged 40 and over. We offered a free volunteer screening program to asymptomatic women for a study using transvaginal ultrasound and color flow Doppler for the detection of pelvic cancer. In the first 2 years, 2117 women were examined, 51.3% post-menopausal. An ovarian cyst was defined as having a maximum diameter of more than 2.4 cm. Color flow was used to identify blood vessels feeding pelvic organs and adnexal enlargements. An abnormal Doppler flow velocity for the ovary was defined as a resistance index of less than 0.41. Ovarian cysts of less than 5 cm with normal Doppler indices were followed up in 6 months to 1 year. An adnexal morphology score was created to quantify the usefulness of this parameter, particularly in postmenopausal women. Indications for surgery were pre-defined as a persistent ovarian cyst of more than 5 cm, a persistent suspicious Doppler and a total endometrial thickness of greater than 0.59 cm in postmenopausal women not taking hormones.A total of 202 women (9.5%) had ovarian cysts. Fourteen women were operated upon because of size criteria, one because of family history and three for persistent abnormal flow. By Doppler study, 15 cysts were predicted to be benign and histology was confirmatory. There were two false positives and one true positive, a stage Ib ovarian cancer. There were no false negatives, although a stage I endometrioid cancer of the ovary was detected 8 months after a negative scan. In those cases in which follow-up data were available, 56% of the cysts regressed in premenopausal women. In postmenopausal women, 28% regressed. Twenty of 1086 postmenopausal women had endometrial biopsies. Three had endometrial cancer, two stage I and one stage IIA. Five had atypical or adenomatous hyperplasia, and seven had benign polyps.So many women have small asymptomatic cysts of the ovary that a major reorientation of physicians' attitudes towards the ovary will have to be introduced to adapt to this new information. In this population, the prevalence of benign epithelial ovarian neoplasms was 7/1000, and of malignancy was 05/1000. In postmenopausal women, the prevalence rate for endometrial cancer was 5/1000, with 5/1000 hyperplasias and 7/1000 benign polyps. When endometrial and ovarian cancer screening are combined, the yield is comparable to that seen in breast and cervical cancer screening.     

Human poisoning with pentachlorophenol and its treatment

A case of intentional intoxication with pentachlorophenol has been described. Salient features observed included pyrexia, diaphoresis, hyperkinesis, muscle twitching, tremors, epigastric tenderness, leg pain, tachypnea, and tachycardia. The patient's restlessness and agitation were controlled with phenytoin and phenobarbital. Forced diuresis with furosemide and mannitol resulted in a large increase in urinary excretion of pentachlorophenol. It is suggested that such therapy may be life saving in such intoxications.     

Morbid obesity treated by gastroplasty: radionuclide gastric emptying studies

Mechanisms by which gastroplasty for morbid obesity causes weight loss are poorly understood. We studied the role of altered gastric emptying in 50 patients before surgery, 1-4 weeks after surgery, and 2-24 months after surgery using technetium-99m pentetate in water for liquid meals and a Tc-99m styrene divinylbenzene copolymer resin in oatmeal for semisolid meals. We determined the emptying half-times of the stomach before and after surgery in the proximal and distal compartments. The proximal compartment emptied promptly in the early and late postoperative periods. The distal compartment emptied liquids at rates similar to those before surgery, while the late postoperative emptying of semisolids was significantly faster. The stoma connecting the two compartments thus permits rapid transit of liquids and semisolids without delay of distal compartment emptying. No correlation was seen between the emptying half-times or changes thereof and eventual weight loss. Delayed gastric emptying is therefore not the mechanism for satiety and weight loss after gastroplasty has been performed.     

Apomorphine-induced alterations in striatal and substantia nigra pars reticulata glutamate following unilateral loss of striatal dopamine

We have reported time-dependent changes in extracellular glutamate within the striatum at 1 and 3 months following a unilateral lesion of the nigrostriatal pathway using the neurotoxin, 6-hydroxydopamine (6-OHDA) (Meshul, C.K., Emre, N., Nakamura, C.M., Allen, C., Donohue, M.K., Buckman, J.F., 1999. Time-dependent changes in striatal glutamate synapses following a 6-hydroxydopamine lesion. Neurosci. 88, 1-16.). The aim of the present study was to determine the effects of such a lesion on glutamate within the substantia nigra pars reticulata (SN-PR) and the effect of subchronic administration of the dopamine D-1/D-2 agonist, apomorphine, on extracellular glutamate within both the striatum and the SN-PR using in vivo microdialysis. One month after the lesion, there is an increase in extracellular glutamate within the striatum and apomorphine treatment leads to a further increase. Within the SN-PR, a loss of striatal dopamine leads to a decrease in extracellular glutamate, while apomorphine treatment leads to a further decrease in nigral glutamate. Three months after a 6-OHDA lesion, there is a decrease in extracellular striatal glutamate, with apomorphine administration leading to essentially no further change in glutamate. The loss of striatal dopamine increased extracellular glutamate within the SN-PR while apomorphine administration resulted in a decrease in extracellular glutamate back to the value observed in the control group. The data suggests that the increase in striatal glutamate 1 month following a 6-OHDA lesion alone or following subchronic apomorphine is consistent with the hypothesis that a decrease in glutamate within the SN-PR leads to activation of the thalamo-cortico-striatal pathway. The decrease in striatal glutamate 3 months after a nigrostriatal lesion is also consistent with the observed increase in extracellular glutamate within the SN-PR, thus leading to a decrease in output of the thalamo-cortico-striatal pathway.     

Characteristics of adolescent street youth with a history of pregnancy

This study examines characteristics of adolescent street youth with histories of pregnancy and documents important factors that merit consideration when providing global sexual health care. STUDY OBJECTIVE: To determine social and behavioral factors associated with a history of pregnancy among adolescent street youth. DESIGN, SETTING, PARTICIPANTS: In a prospective cohort study, female adolescent street youth (14-19 years) ever pregnant (AEP) were compared with adolescents never pregnant (ANP) using data from baseline questionnaires. RESULTS: Among the 225 participants, 41.8% were ever pregnant. Both groups were similar with respect to age (mean 17.8 years) and other socio-economic characteristics. However, AEP were more likely to have been kicked out of home (62.8% vs. 47.3%, P=0.022) and to have run away (78.7% vs. 64.9%, P=0.025) and were homeless younger (mean age: 13.9 vs. 14.7 years, P=0.011) and since a longer period (mean: 4.0 vs. 3.0 years, P=0.001). Both groups had problematic alcohol and drug use: 31.3% had a CAGE score >2; 72.2% had a DAST score >6. Almost half (44.0%) had ever injected drugs and AEP were younger at initiation into drug injection (15.2 years vs. 16.0 years, P=0.049). More AEP had experienced intra-familial or extra-familial sexual abuse (71.3% vs. 56.5%, P=0.024), and had had more than one abuser (71.6% vs. 50.0%, P=0.009). Among those abused by family members, abuse occurred at an earlier age for AEP (mean age: 7.4 vs. 8.9 years, P=0.090) and more AEP reported severe abuse: vaginal penetration (62.2% vs. 26.7%, P=0.004) and anal penetration (29.7% vs. 3.3%, P=0.005). CONCLUSIONS: Histories of severe sexual abuse and early injection drug use are extremely frequent in ever pregnant street adolescents. These factors need to be addressed when planning global health care and sexual health education.     

Analysis of the major histocompatibility complex in graft rejection revisited by gene expression profiles

BACKGROUND: The precise role of major histocompatibility complex (MHC) molecules in graft rejection remains incompletely understood. The important role of foreign peptides in the alloimmune response was recently recognized. METHODS: We performed a comparative study of the functions of minor antigens Class I, Class II, and CD1 in murine cardiac allograft rejection by investigating the expression of a large panel of immune and inflammatory genes. To investigate the role of MHC Class II and I, our protocol analyzed allograft recipients deficient in MHC Class II and b2 microglobulin (b2-M), a critical component of the Class I heterodimer. We also included CD1 deficient recipients to differentiate effects in the beta2-M deficient strain due to CD1 deficiency versus the combined inactivation of CD1 and Class I. The serum cytokines tumor necrosis factor (TNF)-alpha interleukin (IL)-6, interferon (IFN)-gamma and IL-1beta were evaluated posttransplant by ELISA. The intragraft expression of 55 chemokines, chemokine receptors, and CD markers were measured by ribonuclease protection assay. The data were analyzed through hierarchical clustering dendrograms and self-organizing maps. RESULTS: The analysis indicates that each gene deficiency induces both the upregulation and the downregulation of distinct subsets of genes and that similar kinetics of rejection can be attributed to different molecular mechanisms. CONCLUSIONS: The study provides novel insights into the role of classical and non-classical MHC molecules in graft rejection.