Chlamydia trachomatis Subverts Alpha-Actinins To Stabilize Its Inclusion.

Chlamydia trachomatis is the leading cause of sexually transmitted bacterial disease and a global health burden. As an obligate intracellular pathogen, Chlamydia has evolved many strategies to manipulate its host and establish its intracellular niche called the inclusion. C. trachomatis reorganizes the host actin cytoskeleton to form scaffolds around the inclusion and reinforce the growing inclusion membrane. To control the kinetics and formation of actin scaffolds, Chlamydia expresses the effector InaC/CT813, which activates the host GTPase RhoA. Here, we have discovered that InaC stabilizes actin scaffolds through the host actin cross-linking proteins α-actinins 1 and 4. We demonstrate that α-actinins are recruited to the inclusion membrane in an InaC-dependent manner and associate with actin scaffolds that envelop the inclusion. Small interfering RNA (siRNA)-mediated knockdown of α-actinins differentially regulate the frequency of actin scaffolds and impair inclusion stability, leaving them susceptible to rupture and to nonionic detergent extraction. Overall, our data identify new host effectors that are subverted by InaC to stabilize actin scaffolds, highlighting the versatility of InaC as a key regulator of the host cytoskeletal network during Chlamydia infection. IMPORTANCE Despite antibiotics, recurrent C. trachomatis infections cause significant damage to the genital tract in men and women. Without a preventative vaccine, it is paramount to understand the virulence mechanisms that Chlamydia employs to cause disease. In this context, manipulation of the host cytoskeleton is a critical component of Chlamydia development. Actin scaffolds reinforce the integrity of Chlamydia's infectious vacuole, which is a critical barrier between Chlamydia and the host environment. Having previously established that InaC co-opts RhoA to promote the formation of actin scaffolds around the inclusion, we now show that Chlamydia hijacks a new class of host effectors, α-actinins, to cross-link these scaffolds and further stabilize the inclusion. We also establish that a core function of the chlamydial effector InaC is the regulation of cytoskeletal stability during Chlamydia infection. Ultimately, this work expands our understanding of how bacterial pathogens subvert the actin cytoskeleton by targeting fundamental host effector proteins.

Serous retinal detachment in hypertensive posterior reversible encephalopathy syndrome.

In accelerated hypertension, vasogenic brain edema associated with PRES may represent either autoregulatory breakthrough leading to vasodilation or excessive autoregulation leading to vasoconstriction. We describe 2 patients with PRES in accelerated hypertension who had serous retinal detachments, a vasoconstrictive phenomenon. The concurrence of serous retinal detachment and PRES offers intriguing support for the idea that vasoconstriction rather than vasodilation is the mechanism of vasogenic edema in PRES.

Temozolomide (Temodar).

Temozolomide, an oral alkylating agent, is a commonly used medicine in the treatment of anaplastic astrocytoma and glioblastoma multiforme. This paper will present the mechanism of action as well as the clinical role for this chemotherapeutic drug.

Antifungal compounds redirect metabolic pathways in yeasts: metabolites as indicators of modes of action.

AIMS: Metabolic pathways, e.g. biosynthesis of ergosterol or carbohydrate metabolism including respiration, are well-known targets of several fungicides. With our study we wanted to prove that metabolite profiles can be used to classify fungicides according to their mode of action and that concentrations of key metabolites are changed even without detectable reduced growth rates. METHODS AND RESULTS: We exposed the yeasts Candida albicans and Saccharomyces cerevisiae to inhibitors of the electron transport chain and to compounds known to interact with osmotic stress defence pathways. Glycerol and ethanol were chosen as key metabolites of branches of glucose catabolism. Increased glycerol concentrations were observed not only when the osmotic stress response was activated, but also as response to the inhibition of the electron transfer chain, whereas elevated ethanol levels were observed only when the respiratory pathways were blocked. CONCLUSIONS: The treatment of the yeasts Candida albicans and Saccharomyces cerevisiae with antimycotic compounds led to a redirection of metabolic pathways, which could be followed by the quantification of both the metabolites ethanol and glycerol. Only the combination of both concentration profiles allowed the clear distinction between inhibitors of the respiratory chain and effects on the osmotic stress response pathway. IMPACT OF STUDY: The extension of the number of metabolites to a comprehensive quantitative metabolic profile of compound-treated test organisms can be an additional tool in fungicide research allowing the detection of compounds which act on fungi and, moreover, the elucidation of modes of action.

Augmentation of antitumor activity of an antibody-interleukin 2 immunocytokine with chemotherapeutic agents.

PURPOSE: Immune-based therapies, such as the immunocytokine huKS-IL2, exert potent antitumor responses in some animal models by targeting cytokine activity to the tumor microenvironment. We found that certain chemotherapy agents in the appropriate dose and schedule can augment the antitumor activity of huKS-IL2. EXPERIMENTAL DESIGN: Chemotherapy agents were given in a single dose followed 1 day (paclitaxel) or 3 days (cyclophosphamide) later with five daily doses of huKS-IL2 in mice bearing established s.c. tumors, liver metastases, or lung metastases. Tumor models used were CT26/KSA colon, 4T1/KSA mammary, or LLCKSA Lewis lung carcinomas. To measure huKS-IL2 distribution, radiolabeled protein was given to CT26/KSA tumor-bearing mice 1 or 24 h after paclitaxel. huKS-IL2 levels in the tumors were evaluated. RESULTS: Both paclitaxel and cyclophosphamide followed by huKS-IL2 resulted in enhanced antitumor responses compared with either of the treatments alone in the three different tumor models. Results from studies to determine whether the role of the cytotoxic agents in antitumor activity enhancement was related to tumor uptake indicated that a larger fraction of the radiolabeled huKS-IL2 penetrated the tumors when it was administered 24 h after cytotoxic drug "sensitization." CONCLUSION: These data support the idea that prior drug therapy serves to decompress the tumor and lower the diffusion barrier for macromolecules, thus allowing for increased uptake of the huKS-IL2 immunocytokine into the tumor microenvironment. Because the toxicity of the immunocytokine is relatively low at optimal doses, the therapeutic index would likely be greater with the combination treatments.

Improving the efficacy of antibody-interleukin 2 fusion proteins by reducing their interaction with Fc receptors.

Fusion proteins between whole antibodies (Abs) and cytokines (immunocytokines) such as interleukin 2 have shown efficacy in several mouse tumor models despite a circulating half-life that is significantly shorter than that of the original Ab. We have examined the potential mechanisms responsible for clearance and shown that an important factor is enhanced binding to Fc receptor (FcR). Improvements in the half-lives of two different immunocytokines were made by changing the isotype of the human heavy chain C region from IgG1 or IgG3 to those with reduced binding to FcR, e.g., IgG4. The same effect could also be achieved through site-directed mutagenesis of the FcR binding site in the IgG1 H chain. In vitro studies using mouse J774 FcR-expressing cells showed increased binding of interleukin 2-based immunocytokines, relative to their corresponding Abs, and that this was reversed in those fusion proteins made with IgG4 or mutated IgG1 H chains. All of the fusion proteins showing reduced FcR binding also had reduced Ab-dependent cellular cytotoxicity activity, as measured in 4-h chromium release assays. A complete loss of complement-dependent cytotoxicity activity was seen with an IgG4-based immunocytokine derived from an IgG1 Ab with potent activity. Despite these reduced effector functions, the IgG4-based immunocytokines with extended circulating half-lives showed equivalent (in the case of severe combined immunodeficiency mouse xenograft models) or better (in the case of syngeneic models) efficacy in mouse tumor models than the original IgG1-based molecules. These novel immunocytokines may show improved efficacy in therapeutic situations where T cell- rather than natural killer- or complement-mediated antitumor mechanisms are involved.

Antibody-IL-12 fusion proteins are effective in SCID mouse models of prostate and colon carcinoma metastases.

IL-12 is a complex cytokine in both its structure and its range of biologic activities. Fusions of this heterodimeric molecule with an intact antitumor Ab were made to test the feasibility and efficacy of targeting IL-12 to tumors to elicit a local immune response. Fusion proteins composed of the human p35 and p40 subunits had IL-12 bioactivities that were nearly as potent on human immune cells as the rIL-12 standard, but were inactive on mouse cells. Hybrid IL-12 fusion proteins composed of mouse p35 and human p40, fused to Ab, were capable of inducing IFN-gamma, but were much less active on mouse spleen cells than a mouse IL-12 standard. Despite this relatively low activity, the hybrid fusion protein was as effective in a SCID mouse model as a fully active Ab-IL-2 fusion protein in eliminating established pulmonary metastases of CT26 colon carcinoma. Specific targeting of a human IL-12 fusion protein to metastatic prostate carcinoma xenografts was also shown to be effective in SCID mice transplanted with human lymphocyte-activated killer cells. These results demonstrate the importance of directing this potent cytokine to the tumor microenvironment and suggest an important alternative to systemic IL-12 administration or gene therapy for increasing its therapeutic index.

Second-look laparotomy in advanced ovarian cancer: a critical assessment of morbidity and impact on survival.

BACKGROUND: The routine inclusion of second-look laparotomy in the management of patients with epithelial ovarian cancer is controversial. At issue is the justification of morbidity incumbent upon surgery and the possible survival benefit of secondary cytoreduction. METHODS: The rate of major complications of surgery was assessed among 100 consecutive patients with FIGO stage III or IV epithelial ovarian cancer who underwent second-look laparotomy. All patients demonstrated a complete clinical and biochemical (CA125 < 35 U/ml) response to first-line therapy. Patients were stratified based on findings at surgery. Patients in group 1 (n = 37) had a negative second-look laparotomy. Patients in group 2 (n = 35) had only microscopically appreciable disease. Patients in group 3 (n = 28) had macroscopic disease. Cytoreductive efforts aimed at resection of all macroscopic disease were carried out for patients in group 3. RESULTS: Thirteen patients (13%) had 15 major complications at surgery. Comparison of the complication rates for patients in groups 1, 2, and 3, of 10%, 8.5%, and 21.4%, respectively, did not achieve statistical significance (p = 0.228). The estimated 5-year survival for patients in groups 1, 2, and 3 of 63.9%, 39.8%, and 14.2%, did differ significantly (p < 0.0001). Cytoreductive efforts resulted in the resection of all macroscopic disease in 18 of 28 patients (64.2%) in group 3. The median survival for this group of 18 patients was 33 months, and estimated 5-year survival was 20%. These values do not differ significantly from those observed for patients in group 2. CONCLUSION: The major complication rate associated with second-look laparotomy is not prohibitive Secondary cytoreductive efforts may result in improved survival for patients with epithelial ovarian cancer.

FP-21399 blocks HIV envelope protein-mediated membrane fusion and concentrates in lymph nodes.

The identification of fusin and other chemokine receptors as coreceptors for HIV-1 has renewed the interest in agents that may prevent viral entry. Polyanionic compounds such as dextran sulfate, curdian sulfate, and suramin act on the V3 loop of the viral envelope and may prevent its interaction with fusin. These agents show activity against a wide range of HIV-1 strains, but have undesirable circulating half-life, bioavailability, and toxicity. We have developed a small molecule inhibitor of HIV-1 that has several advantages over these other agents. FP-21399 is a novel compound of the bis(disulfonaphthalene) dimethoxybenzene class that blocks entry of HIV into CD4+ cells and blocks fusion of infected and noninfected CD4+ cells. This compound only weakly inhibits binding of CD4 and gp120, at concentrations much greater than are required to block viral entry. Furthermore, FP-21399 can block the interaction between gp120 and antibodies directed against the V3 loop, but does not block binding of antibodies directed against the V4 loop. Animal studies demonstrate that FP-21399 is concentrated in lymph nodes, making it a promising compound for anti-HIV therapy. In SCID mice reconstituted with human immune cells, maintenance of HIV-1 infection was blocked by a 5-day treatment with low doses of FP-21399, suggesting that lymph node accumulation may contribute to antiviral activity. Finally, attempts to generate drug-resistant virus in cell culture resulted in only weakly resistant variants with IC90 values that are much lower than concentrations of FP-21399 found in lymph nodes.

Relation between residential radon concentrations and housing characteristics. The Cracow Study.

The survey on indoor radon exposure was undertaken to explain whether the excess in lung cancer deaths in Cracow city center may be attributed to this particular exposure. A total of 310 detectors was placed in households randomly chosen from three homogenous strata of residential buildings. The first stratum included house in the old city center constructed predominantly out of the stone bricks. The second stratum covered area of the city with big apartment condominiums built out of concrete blocks. The third stratum consisted of single family houses located in a suburban area. From each of these residency strata a random sample of equal number of households has been chosen and the radon detectors were placed in households located at different levels of buildings. The three-month radon sampling data were used to determine the distribution of various levels of radon in the households. In the measurement of radon exposure the Landauer alpha-track samplers were used. The data collected show that the best single predictor of indoor radon concentrations was type of building. Other variables found to be associated significantly with indoor concentrations were household level in the building and house age. In general, residences with a concrete slabs and dwellings with rarely-opened windows were found to have slightly higher radon concentrations.

Role of inter-heavy and light chain disulfide bonds in the effector functions of human immunoglobulin IgG1.

The role of inter-heavy and light chain disulfide bonds in the effector functions of human IgG1 was investigated. This was accomplished by mutating appropriate sites in IgG1 such that the disulfide bond pattern now resembled that of IgG4. The effector functions of the mutant antibody were then compared to native IgG1 and IgG4. The antibody-dependent cell cytotoxicity activity was completely abolished in the mutant and the complement-dependent cytotoxicity assay was reduced fifteen-fold. The results suggest that the inter-heavy and light chain disulfide bond pattern of an antibody molecule play a role in its effector functions.

Human exposure to pollutants in Poland.

Serious environmental problems caused by decades of mismanagement of Poland's natural resources have been brought to light in recent years. All environmental media--air, water, food, and soil--have been burdened with toxic chemicals. Some environmental problems are so severe that the sources of pollution and the mitigation techniques needed are obvious, requiring no further research, but rather common sense, monies, and determination to implement the necessary controls and mitigation procedures. This paper will not address these obvious cases. Rather it will address that spectrum of environmental problems which requires a better understanding of public health risk in order to develop effective risk management strategies. Because these problems are numerous and monies limited, policy makers will need to set priorities both for research projects and control options. Using environmental concentration data presently available from Poland (especially for air), the paper will estimate human exposures, will point out research and monitoring needs, and hopefully, will lend credence to the concept that environmental policies and risk reduction strategies will be most effective if the Total Human Exposure Concept is used as the guiding scientific principle in risk assessment and management programs.

[Anticipated results of arterial surgery in chronic atherosclerotic ischemia of the lower limb]. / Przewidywanie wyników operacji naprawczych tetnic w przewleklym miazdzycowym niedokrwieniu konczyn dolnych.

In 1983-1988, the results of conservative and surgical treatment of patients with atherosclerotic ischemia of the lower limbs were assessed with the aid of a questionnaire. A possibility of prognosis was assessed with the use of mathematically processed data obtained with such approach. An effect of clinical symptoms (intermittent claudication distance, resting pain, necrosis) and stage of the disease (duration, K/R index) and risk factors (blood cholesterol, triglycerides, diabetes mellitus, ischemic heart disease, arterial hypertension) on the result of surgical treatment was analysed. The obtained results suggest that clinical symptoms and risk factors may predict the results of surgical treatment in the atherosclerotic ischemia of the lower limbs.

Irritant effects of formaldehyde exposure in mobile homes.

This paper reports the irritant effects associated with formaldehyde exposures in mobile homes. Week-long, integrated formaldehyde concentrations were measured using passive monitors in summer and winter while the mobile home residents continued their normal activities. Information on acute health problems, chronic respiratory/allergic illnesses, smoking behavior, demographic variables, and time spent at home was obtained on over 1000 individuals during the sampling period. Measured formaldehyde concentrations varied from under the limit of detection (0.01 ppm) to 0.46 ppm. Formaldehyde exposure was estimated for each individual by multiplying the concentration measured in his or her home by the time he or she spent at home. Irritant effects were found to be associated with formaldehyde exposure after controlling for age, sex, smoking status, and chronic illnesses using a logistic procedure. Some of the interaction terms found to be significant indicated that there were synergistic effects between formaldehyde exposure and chronic health problems.

Seasonal variations and trends in concentrations of filter-collected polycyclic aromatic hydrocarbons (PAH) and mutagenic activity in the San Francisco Bay area.

Air monitoring in the San Francisco Bay Area was carried out to measure outdoor community air concentrations of polycyclic aromatic hydrocarbons (PAH) and mutagenic activity (mutagenicity) in particulate organic matter (POM). Monitoring began in 1979 and is currently conducted at six stations. PAH and mutagenicity tests were performed on organic extracts prepared from high volume (hi-vol) filters composited every four months, by meterological season. PAH were determined by high pressure liquid chromatography (HPLC) with fluorescence and ultraviolet detection. Mutagenicity was measured in the Ames Salmonella bioassay using strain TA98 with and without metabolic activation. The nine-year mean concentration of benzo(a)pyrene (BaP) was 0.4 ng/m3. The mutagenicity of this amount of BaP accounted for only about 0.2% of the observed mutagenicity in POM and other measured PAH accounted for even less. Concentrations of PAH and mutagenicity were three to nine times higher during the winter than during other seasons. Year-to-year wintertime trends in several PAH were also seen. Early in the 1980s, winter concentrations of BaP and benzo (g,h,i)perylene increased. However since the mid-1980's, their concentrations have fallen. The decrease in PAH concentrations may be the result of an increasing proportion of vehicles with relatively low organic emissions. In contrast to PAH, mutagenicity did not show significantly year-to-year time trends.

Targeting human cytotoxic T lymphocytes to kill heterologous epidermal growth factor receptor-bearing tumor cells. Tumor-infiltrating lymphocyte/hormone receptor/recombinant antibody.

A genetically engineered conjugate between an anti-CD3 antibody and epidermal growth factor (EGF) was tested for its ability to mediate the lysis of receptor-bearing cells by human CTL. This construct was made by fusing an EGF coding sequence to the 3' end of the human gamma-1 H chain gene sequence and expressing the modified gene in transfected cells together with the V regions of a mouse antibody specific for the human T cell marker, CD3. The resulting conjugate was able to compete with EGF for its receptor and, at extremely low concentrations, was able to mediate the lysis of receptor-bearing tumor targets by a tumor-infiltrating lymphocytes line or by a CTL line established from peripheral blood. The construction of such conjugates by genetic engineering represents a general approach to the direct expression of highly specific hetero-bifunctional reagents without the necessity of further in vitro manipulations.

The relation between policy and research planning.

The crucial role of total exposure assessment (TEA) in environmental epidemiology, risk assessment, risk management, and disease diagnosis and treatment has been appreciated only in recent years, and then by a relatively small number of policy makers and scientists. Thus TEA is a relatively new research field with a limited data base. As a result, research needs cover a broad range. However, setting research priorities with limited resources is a problem. Agencies usually develop criteria to set priorities. The criteria often have at least one element in common, namely, that the research be compatible with policy goals. However, policies are developed, at least in part, using research data, thus making policy and research planning an iterative process. TEA, by definition, is related to multi-media and multiple routes of exposure. Because of this, research planning can be hampered by differences in policies of programs dealing with different media and routes of exposure. Thus it is important to develop better ways to facilitate communication among policy makers, research planners, and researchers. This paper will argue that one such way is the proper use of the basic scientific tenet of hypothesis testing in planning, executing, and evaluating research. The paper will discuss this tenet, and recommend that exposure researchers use it, not only to improve the quality of their individual research but also to provide a better mechanism for setting priorities for research projects.

The use of microcapsules for high density growth of human tumor infiltrating lymphocytes and other immune reactive T cells.

The use of microcapsules to achieve high density growth of tumor infiltrating lymphocytes (TIL) and other antigen-specific human T cells is described. Whereas human T cells in suspension cultures usually do not exceed 1-2 x 10(6) cells/ml, densities approaching that found in living tissues (greater than 10(8) cells/ml) have been observed for microcapsule cultures. TIL and human peripheral blood-derived T cells can be routinely recovered from microcapsules with viabilities greater than or equal to 90%. The recovered cells retain their antigen reactivities and bear cell surface phenotypes identical to their counterparts grown in suspension culture. These findings suggest that microcapsule technology could prove valuable in generating the vast numbers of cells required for TIL therapy and other forms of adoptive immunotherapy with T cells.

Antigen binding and biological activities of engineered mutant chimeric antibodies with human tumor specificities.

The constant region of the human gamma 1 chain was mutated either by deleting the second domain (CH2) or by mutating the two hinge region cysteine residues, normally involved in the inter-heavy chain disulfide bond formation, to serines. The effects of these mutations on chain assembly, antigen binding, complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) were measured after expressing the human constant regions together with mouse variable regions encoding anti-tumor cell specificities. The CH2-deleted chimeric antibody was found to have increased antigen binding activity and little (ADCC) or no (CDC) biological activity. The cysteine to serine hinge region mutant antibody had normal or slightly reduced antigen binding activity, greatly reduced ADCC activity, and a reduced, but still significant, ability to mediate CDC. These results reflect the complexity of the interactions between the immunoglobulin domains and their role in balancing the antigen binding and effector functions of antibodies. They suggest further that such antibodies may be useful in applications, such as the in vivo imaging of tumors, where the loss of effector function (e.g., Fc receptor binding) is desired.