Effective antigen cross-presentation by prostate cancer patients' dendritic cells: implications for prostate cancer immunotherapy.

Despite the potency with which dendritic cells (DCs) are able to utilize the exogenous MHC I antigen cross-presentation pathway to cross-present antigen for the activation of killer T cells in model systems, concern about defects in immune function in cancer patients has led to uncertainty regarding whether immune cells derived from patients can effectively be used to generate tumor vaccines. We have undertaken a careful analysis of the potency of using DCs obtained from prostate cancer patients to cross-present antigen derived from human prostate tumor cells for the activation of antigen-specific T cells. Such DCs can be matured ex vivo into functionally active cells and are capable of cross-presenting influenza antigen derived from internalized apoptotic prostate tumor cells. Importantly, we demonstrate effective stimulation of both CD4+ and CD8+ T cells, as evident by production of IFN-gamma, and the ability of CD8+ T cells to differentiate into effector CTLs. These results, defining conditions in which prostate cancer patient DCs can efficiently utilize the cross-presentation pathway and in which apoptotic tumor can serve as a source of antigen for DCs to activate T cells, demonstrate that this system warrants clinical study as a potential immunotherapy.

Dendritic cell maturation is required for the cross-tolerization of CD8+ T cells.

In vivo models have shown that tissue-restricted antigen may be captured by bone marrow-derived cells and cross-presented for the tolerization of CD8+ T cells. Although these studies have shown peripheral tolerization of CD8+ T cells, the mechanism of antigen transfer and the nature of the antigen-presenting cell (APC) remain undefined. We report here the establishment of an in vitro system for the study of cross-tolerance and show that dendritic cells (DCs) phagocytose apoptotic cells and tolerize antigen-specific CD8+ T cells when cognate CD4+ T helper cells are absent. Using this system, we directly tested the "two-signal" hypothesis for the regulation of priming versus tolerance. We found that the same CD83+ myeloid-derived DCs were required for both cross-priming and cross-tolerance. These data suggested that the current model for peripheral T cell tolerance, "signal 1 in the absence of signal 2", requires refinement: the critical checkpoint is not DC maturation, but instead the presence of a third signal, which is active at the DC-CD4+ T cell interface.

Hematopoietic expression of HOXB4 is regulated in normal and leukemic stem cells through transcriptional activation of the HOXB4 promoter by upstream stimulating factor (USF)-1 and USF-2.

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34(+) cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.

Deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: clinical and pathological characteristics of the disease. For the Outbreak Study Group.

From April through June 1997, 29 previously healthy children aged <6 years (median, 1.5 years) in Sarawak, Malaysia, died of rapidly progressive cardiorespiratory failure during an outbreak of hand, foot, and mouth disease caused primarily by enterovirus 71 (EV71). The case children were hospitalized after a short illness (median duration, 2 days) that usually included fever (in 100% of case children), oral ulcers (66%), and extremity rashes (62%). The illness rapidly progressed to include seizures (28%), flaccid limb weakness (17%), or cardiopulmonary symptoms (of 24 children, 17 had chest radiographs showing pulmonary edema, and 24 had echocardiograms showing left ventricular dysfunction), resulting in cardiopulmonary arrest soon after hospitalization (median time, 9 h). Cardiac tissue from 10 patients showed normal myocardium, but central nervous system tissue from 5 patients showed inflammatory changes. Brain-stem specimens from 2 patients were available, and both specimens showed extensive neuronal degeneration, inflammation, and necrosis, suggesting that a central nervous system infection was responsible for the disease, with the cardiopulmonary dysfunction being neurogenic in origin. EV71 and possibly an adenovirus, other enteroviruses, or unknown cofactors are likely responsible for this rapidly fatal disease.

One-step 2-minute test to detect typhoid-specific antibodies based on particle separation in tubes.

Typhoid fever is caused by Salmonella typhi. Detection of anti-S. typhi antibodies in the patient is a useful diagnostic aid. Among the various methods developed over the years for this purpose, the Widal test, based on bacterial agglutination, has remained the most widely used, even though it is neither specific nor sensitive. Its popularity stems from the fact that it is simple to use and inexpensive. We describe a new test which also uses a simple one-step procedure but is more rapid and accurate than the Widal. The new test (TUBEX) detects anti-Salmonella O9 (both immunoglobulin M [IgM] and IgG) antibodies in patients by inhibiting the binding between an anti-O9 IgM monoclonal antibody (MAb) conjugated to colored latex particles and S. typhi lipopolysaccharide (LPS) conjugated to magnetic latex particles. The reactants are mixed in a specially designed microtube for 2 min, and the result is read based on the resultant color of the supernatant following forced sedimentation of the magnetic beads. In the absence of inhibitory antibodies, there is a color change (from blue to red) due to cosedimentation of the indicator particles with the magnetic particles, whereas if these antibodies are present, they prevent such a change to a degree dependent on their concentration. Preliminary examination of TUBEX using the anti-O9 MAb and irrelevant MAbs as inhibitors revealed the test to be specific and reproducible, with an analytical sensitivity of 16 micrograms per ml of antibody. The reagents remained stable for at least 9 months when kept at 4 degrees C. In the examination of 16 stored sera obtained from 14 patients with proven cases of typhoid fever and 78 serum samples from 75 subjects without typhoid fever, TUBEX was found to be 100% sensitive and 100% specific. The nontyphoid group comprised 26 healthy blood donors, 30 antinuclear antibody (ANA)-negative patients, 9 ANA-positive patients, of whom 1 was positive for anti-DNA antibody, 4 typhus patients, and 6 septicemic patients. In addition, the sera obtained from 11 patients clinically diagnosed as having typhoid fever were all positive in the test. The TUBEX results correlated to some extent, albeit insignificantly (r = 0.38, P = 0.07), with those of an enzyme-linked immunoassay (ELISA) which used a similar detection format (inhibition) and reagents (S. typhi LPS and anti-O9 antibody). TUBEX correlated very well with ELISAs which detected anti-S. typhi LPS IgM (r = 0.58, P = 0.003) or IgG (r = 0.54, P = 0.006) antibodies from the typhoid patients. There was no correlation with the Widal test. The TUBEX test, if performed on slides (instead of tubes) or with soluble antigen (instead of antigen-conjugated magnetic beads), suffered significantly in sensitivity. Direct agglutination tests using LPS-conjugated indicator particles performed either on slides or in microwells also failed to detect antibodies from the majority of typhoid patients. Thus, TUBEX appears to be well designed and well suited for use in the laboratory or by the bedside as a simple, rapid aid to the routine diagnosis of typhoid fever.

Pulsed-field gel electrophoresis as an epidemiologic tool in the investigation of laboratory acquired Salmonella typhi infection.

Strains of Salmonella typhi implicated in two separate cases of laboratory acquired infection from patients and the medical laboratory technologists who processed the patients' samples were analysed by pulsed-field gel electrophoresis. Although all four isolates were of bacteriophage type E1, PFGE was able to demonstrate that the strains responsible for the two laboratory acquired cases were not genetically related. The PFGE patterns of the isolates from the MLTs were found to be identical to those of the corresponding patients after digestion with restriction enzyme AvrII. This provided genetic as well as epidemiological evidence for the source of the laboratory acquired infections.

Salmonella serotypes isolated in Malaysia over the ten-year period 1983-1992.

From January 1983 to December 1992 a total of 20,874 salmonella were serotyped in the Bacteriology Division IMR, which showed an increase of 100% compared to the previous ten-years. There were 97 serotypes which belonged to 22 Kauffmann-white groups. Twenty two serotypes hitherto were seen in this study period. S. typhi was the commonest serotype isolated. Overall there was a rise in the isolation of non-typhoidal salmonella particularly S. enteritidis which increased by 760% and S. blockley which increased by 720%. However there is a drop in the isolation of S typhimurium by 223% and S. paratyphi B by 319%.

Bacterial contamination of hospital disinfectants.

A study to determine contamination of diluted disinfectants at different points in preparation and use in 6 Malaysian hospitals was done using the in-use test. A growth of > or = 250 organisms/ml was taken as an indication of contamination. A total of 342 (7.9%) of the 4316 freshly diluted samples collected from disinfectant bottles in the pharmacy were found to be contaminated. The bacterial isolates obtained were Pseudomonas spp. (42.4%), Moraxella spp. (22.0%), Flavobacterium spp. (11.6%) and Enterobacter spp. (4.2%). Three hundred and sixty seven out of 2278 ward stock were contaminated. The isolates were Pseudomonas spp. (48.6%), Moraxella spp. (17.8%), Acinetobacter spp. (8.9%) and Flavobacterium spp. (7.0%). Of the 9265 disinfectants in-use, 1519 (16.4%) were contaminated. Among the organisms isolated were Pseudomonas spp. (44.3%), Bacillus spp. (13.0%), Enterobacter spp. (9.5%) and Acinetobacter spp. (7.3%). The results indicated a high level of contamination of diluted disinfectants prepared in the pharmacy, stored and used in the wards. This gave a high index of suspicion that recommendations for cleaning of disinfectant containers before refilling, handling of diluted stock solutions and using of disinfectants were not closely adhered to. Standard disinfection procedures outlined in the disinfection and sterilization policy by the Ministry of Health should therefore be followed.

Disinfection: gaps between recommended and actual practice.

Basic practices on disinfection was surveyed in 6 hospitals using an observation and interview checklist. Two surveys were done, one pre-(first survey) and one post-intervention (second survey). The disinfection and sterilization policy of the Ministry of Health was not available in 66 (70.2%) and 12 (13%) of the units in the first and second survey respectively. In the second survey, staff in all the units washed disinfectant containers before refilling compared with 41.5% of the units in the first survey. Dilution of disinfectants not recommended was found to be used in the first survey. Storing cleaned and sterile items in disinfectants, using disinfectant as a substitute for sterilization of autoclavable items and not decontaminating spillages were some of the wrong practices observed. Considerable improvements were made in the second survey. Improper usage of disinfectants was also indicated by failure of the in-use test. Rate of failure of disinfectants in-use decreased from 11.6% in the first survey to 5.0% in the second survey. To ensure proper disinfection practices, a comprehensive training program on disinfection is required for nurses and attendants.

An evaluation of knowledge and awareness of disinfection and sterilization among health care workers.

Awareness of the disinfection and sterilization policy among hospital staff and their knowledge in basic principles and methods of disinfection and sterilization were studied before and after intervention using a self-administered questionnaire. Survey results showed that awareness (56.2%) before intervention was unsatisfactory. The nurses were more aware of the policy than other groups of medical personnel. Those unaware of the policy perform duties from memory or verbal instructions. A significant increase in awareness to 73.3% was observed after intervention (p < 0.05). Knowledge on methods of decontamination, disinfection and sterilization of equipment varies widely from 28.8% to 90.1%. 23.1% were unaware of the temperature used for sterilization while 72.4% did not know how containers of disinfectant should be refill. Only 14.7% knew the recommended method for washing containers. With education improvement was observed. The average knowledge improved from 44.4% to 57.3%. Our results indicated that continuous in-service education is needed to improve, supplement and update knowledge in this field after basic training. In addition orientation programs for new staff should also be aimed at creating awareness and providing information on guidelines and policies related to their duties.

Antimicrobial resistance pattern of bacteria isolated from patients seen by private practitioners in the Klang Valley.

Data on bacterial resistance in patients seen by general practitioners are usually not readily available. The objective of this paper is to present the antimicrobial resistance pattern of bacteria isolated from patients seen by private practitioners in the Klang Valley. A total of 18 clinics participated in this study. From mid August 1991 to end of June 1993, 2,823 specimens were received. Throat swabs and urine specimens constituted 56% of all the specimens. A large proportion of the specimens (55%) yielded no growth or just normal flora. The common bacteria encountered were Staphylococcus aureus (18.4%), Escherichia coli (16.2%), Klebsiella spp (13.7%) and Neisseria gonorrhoeae (9.3%). The S. aureus strains were mainly isolated from wound, pus and ear swabs. Not one out of the 218 strains tested was resistant to methicillin. In vitro susceptibility tests showed that 91% were resistant to penicillin while 23% were resistant to tetracycline and 13% to erythromycin. Eighty-two percent of the E. coli were isolated from urine. It was also the most common isolate from urine. Fifty percent of these strains were resistant to ampicillin, 33% to cotrimoxazole, 17% to cephalothin, 21% to ampicillin-sulbactam, 18% to amoxycillin-clavulanic acid while only 2.3% were resistant to nalidixic acid and nitrofurantoin and none to cefuroxime. Generally the gram negative bacilli encountered in general practice are less resistant to the third generation cephalosporins and aminoglycosides when compared to the hospital strains.

Antimicrobial resistance in 6 Malaysian general hospitals.

Knowledge of local antimicrobial resistance patterns of bacteria is a valuable guide to empirical antimicrobial therapy. This paper reports the resistance patterns of more than 36,000 bacteria isolated between August 1991 and July 1992 in six Malaysian hospitals and discusses the implications of the results. A customized menu driven software programme was developed to analyse the results. Generally, resistance to the commonly used antibiotics like ampicillin, cloxacillin, cephalosporins, gentamicin, cotrimoxazole and tetracycline was high. Some differences in resistance rate amongst the six hospitals were also noted. Continuous surveillance of antimicrobial resistance in hospitals is encouraged for the effective control of the emergence of antimicrobial resistance.

Identification of a human group C rotavirus in Malaysia.

Stool specimens from 334 infants and young children hospitalized with diarrhea in the General Hospital, Kuala Lumpur, Malaysia between August and November, 1987 were analyzed for the presence of rotavirus double-stranded (ds) RNA by polyacrylamide gel electrophoresis. Of the 334 specimens analyzed, 32 (9.6%) were positive for rotavirus RNA. One specimen (designated G147) exhibited a ds RNA electropherotype profile characteristic of Group C rotavirus and was selected for further characterization. In Northern blot hybridization studies, the gene 5 segment of strain G147 hybridized with a cDNA probe generated from the cloned gene 5 (which encodes the VP6 inner capsid protein that is group specific) of porcine Group C rotavirus strain Cowden, confirming the classification of strain G147 in Group C. The association of Group C rotavirus with diarrheal illness in Malaysia is consistent with earlier studies that suggest a global distribution of this virus and supports the need for additional epidemiologic studies.

Epidemiology and control of malaria in Malaysia.

In spite of more than 30 years of control activities, malaria continues to be the most important parasitic infection in Malaysia, accounting for 39,189 confirmed cases in 1991, giving an annual parasite incidence rate of 2.2 per 1,000 population. Some factors contributing to the continued transmission of malaria are the development of drug resistant Plasmodium falciparum, changes in vector behavior, and ecological changes due to socio-economic reasons. Malaria parasite rates are higher among the Aborigines, land scheme settlers and those in intimate contact with the jungle, like loggers. There has been no substantial change in the proportion of the three common malaria species responsible for infections, P. falciparum, P. vivax, P. malariae and mixed infections accounting for about 70%, 28%, 1% and 1%, respectively of all infections. Drug resistant P. falciparum is unevenly distributed in Malaysia, but based on clinical experience and in vitro drug sensitivity studies, chloroquine resistance is frequently encountered. There has been clinical and laboratory evidence of resistance to sulfadoxine/pyrimethamine combination as well as quinine, but all these have so far been successfully treated with a combination of quinine and tetracycline. The eradication of the disease is impossible in the near future but there is confidence that with better surveillance techniques and the use of alternative control measures like permethrin impregnated bed-nets to complement existing ones, the target of bringing down the annual parasite incidence to 2 per 1,000 population during the Sixth Malaysian Plan period (1991-1995) can be achieved.

A one year community-based study on the incidence of diarrhoea and rotavirus infection in urban and suburban Malaysian children.

A 1 year longitudinal study of 156 Malaysian children from urban and suburban areas in the Klang Valley revealed that the incidence rate of diarrhoea was 23.6 per 100 person-year with abnormal faeces reported on 0.26% of the total days of observation. Diarrhoea cases were detected in children from all socioeconomic classes. Rotavirus was isolated from 12% of the diarrheic children and asymptomatic rotavirus infection occurred in 3.2% of the children. All rotaviruses isolated were group A rotaviruses with long electrophoretypic pattern.

Dietary palmitic and oleic acids exert similar effects on serum cholesterol and lipoprotein profiles in normocholesterolemic men and women.

To compare the effects of dietary palmitic acid (16:0) vs oleic acid (18:1) on serum lipids, lipoproteins, and plasma eicosanoids, 33 normocholesterolemic subjects (20 males, 13 females; ages 22-41 years) were challenged with a coconut oil-rich diet for 4 weeks. Subsequently they were assigned to either a palm olein-rich or olive oil-rich diet followed by a dietary crossover during two consecutive 6-week periods. Each test oil served as the sole cooking oil and contributed 23% of dietary energy or two-thirds of the total daily fat intake. Dietary myristic acid (14:0) and lauric acid (12:0) from coconut oil significantly raised all the serum lipid and lipoprotein parameters measured. Subsequent one-to-one exchange of 7% energy between 16:0 (palm olein diet) and 18:1 (olive oil diet) resulted in identical serum total cholesterol (192, 193 mg/dl), low-density lipoprotein cholesterol (LDL-C) (130, 131 mg/dl), high-density lipoprotein cholesterol (HDL-C) (41, 42 mg/dl), and triglyceride (TG) (108, 106 mg/dl) concentrations. Effects attributed to gender included higher HDL in females and higher TG in males associated with the tendency for higher LDL and LDL/HDL ratios in men. However, both sexes were equally responsive to changes in dietary fat saturation. The results indicate that in healthy, normocholesterolemic humans, dietary 16:0 can be exchanged for 18:1 within the range of these fatty acids normally present in typical diets without affecting the serum lipoprotein cholesterol concentration or distribution. In addition, replacement of 12:0 + 14:0 by 16:0 + 18:1, but especially 16:0 or some component of palm olein, appeared to have a beneficial impact on an important index of thrombogenesis, i.e., the thromboxane/prostacyclin ratio in plasma.

Significance of circumsporozoite-specific antibody in the natural transmission of Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae in an aboriginal (Orang Asli) population of central peninsula Malaysia.

Two hundred and seventy-five Orang Asli volunteers living in nine villages in the Pos Legap Valley of Perak State, peninsular Malaysia, participated in a prospective study designed to characterize the epidemiological, parasitological, and entomological characteristics of Plasmodium falciparum, P. vivax, and P. malariae malaria transmission. Prevalence rates for the three plasmodial species at initiation of the study ranged from 56% in the 0-4-year-old age group to 0% in individuals over the age of 40. Entomological surveys were conducted, enabling us to determine mosquito salivary gland-positive rates and entomological inoculation rates of 1.2 infectious mosquito bites per person per month for P. falciparum, 2.4 for P. vivax, and 0.3 for P. malariae. Cumulative incidence rates over the 16 weeks of the study, following radical cure of all volunteers, were 22.5% for P. falciparum, 12.7% for P. vivax, and 1.5% for P. malariae. The median baseline antibody titer against the immunodominant repetitive B cell epitope of P. falciparum or P. vivax circumsporozoite protein was significantly higher for volunteers who did not become parasitemic. Volunteers were selected for further study if they had evidence of being challenged with P. falciparum sporozoites during the study, based on a two-fold or greater increase in antibody titer against the immunodominant repetitive B cell epitope of the circumsporozoite protein. Resistance to infection was seen in six of 10 individuals who had high (greater than 25 OD units) baseline ELISA titers, compared with only three of 24 individuals who had low baseline ELISA titers (chi 2 P less than 0.02). A similar analysis for P. vivax did not show a significant correlation.