Postchemotherapy and tumor-selective targeting with the La-specific DAB4 monoclonal antibody relates to apoptotic cell clearance

Early identification of tumor responses to treatment is crucial for devising more effective and safer cancer treatments. No widely applicable, noninvasive method currently exists for specifically detecting tumor cell death after cytotoxic treatment and thus for predicting treatment outcomes.

Development of monoclonal antibodies against Vibrio pathogens

Monoclonal antibodies were developed against pathogenic vibrios for use in rapid identification in disease situations of humans, fish and shellfish. Of the 12 fusions performed using V. alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus and V. vulnificus, a total of 102 hybridomas were obtained. Based on cross-reactivity of a wide range of Vibrio strains and other gram-negative bacteria, three broad types of monoclonal antibodies were found. The three categories were: (1) ones that were species-specific or specific to a particular surface antigen, (2) a large number that reacted with several Vibrio species, and (3) three that reacted with most Vibrio strains but no other gram-negative bacteria. Each species-specific monoclonal antibody only recognized its corresponding Vibrio species and was used for identifying unknown species, confirming diagnosis of clinical isolates. In addition, several monoclonal antibodies only cross-reacted with similar Vibrio species, e.g. V. parahaemolyticus and V. alginolyticus which share a common H-antigen. Monoclonal antibodies reacting with several Vibrio species were not of particular use in diagnostic situations. Three monoclonal antibodies of the last group did not react with other genera of the family Vibrionaceae, namely Aeromonas, Photobacterium and Plesiomonas nor a wide range of gram-negative enteric bacteria. These data indicated the existence of an antigenic surface determinant common to Vibrio species. One monoclonal reacted with the heat-stable antigenic determinants on the cell surface as v as lipopolysaccharide extracted from all the vibrios studied, thus making it useful for large- scale screening of acute infections of vibrios. In a blind test, seven Vibrio species, isolated from 6 marine and a freshwater source were identified by two laboratories using phenetic tests. Results of immunotyping using monoclonals, three of seven were diagnosed as the same species, another three were designated as Vibrio species but could not be classified further due to the library not having the corresponding monoclonal, and one was diagnostically questionable. Two further tests were carried out. An unknown Vibrio formalin-fixed isolated from diseased marine animal was identified as V. parahaemolyticus by ELISA and FITC. Clinical human isolates of V. alginolyticus, V. parahaemolyticus and V. vulnificus were confirmed by monoclonals. Australian isolates of V. anguillarum appeared to be mostly of serotype O1. monoclonals raised to V. anguillarum AFHRL 1 reacted with only serotype O1 from Denmark but also most Australian isolates. All vibrios pathogenic to fish and shellfish, i.e. V. anguillarum, V. ordalii, V. alginolyticus, V. carchariae, V. cholerae, V. damsela, V. harveyi, V. parahaemolyticus and V. vulnificus, were used for attachment studies to fish cells using phase contrast and FITC-immunofluorescence microscopy. Of these vibrios, V. anguillarum, V. ordalii and V. perahaemolyticus, were found to adhere to different cells and tissues of rainbow trout while others did not appear to attach. However, attachment was inhibited by monoclonal antibodies specific to only these three vibrios. Lipopolysaccharide is well known as being a contributing factor in pathogenicity of gram-negative bacteria. PAGE electrophoresis of extracted LPS from 9 strains covering 6 Vibrio species showed the presence of a common 15,000 D fragment. This fragment was verified by immunoblotting with a genus-specific monoclonal antibody (i.e. F11P411F) recognizing nearly all vibrios. The common LPS fragment was separated and used to raise polyclonal antisera in mouse which reacted strongly with LPS itself, live as well as sodium azide-killed vibrios, but not with other gram-negative bacteria. This raised the possibility of developing vaccine from Vibrio LPS. Monoclonal antibodies developed in the present study enabled rapid identification of a number of pathogenic Vibrio species. There is still further work to produce monoclonal antibodies against additional vibrios that are probably pathogenic. These included V. fluvialis, V. hollisae, V. metschnikovii, V. minicus, V. salmonella and V. tubiashii. Together the application will be of significance in clinical diagnostic work, in the monitoring of vibriosis in fish farms and in quarantine.

CD1d-restricted NKT cells contribute to malarial splenomegaly and enhance parasite-specific antibody responses

CD1d-restricted NKT cells are a novel T cell lineage with unusual features. They co-express some NK cell receptors and recognize glycolipid antigens through an invariant T cell receptor (TCR) in the context of CD1d molecules. Upon activation through the TCR, NKT cells produce large amounts of IFN- and IL-4. It has been proposed that rapid cytokine output by activated NKT cells may induce bystander activation of other lymphoid lineages. The impact of CD1d-restricted NKT cell activation in the induction of B cell-mediated immune responses to infection is still unclear. We show here that CD1-restricted NKT cells contribute to malarial splenomegaly associated with expansion of the splenic B cell pool and enhance parasite-specific antibody formation in response to Plasmodium berghei infection. The increased B cell-mediated response correlates with the ability of NKT cells to promote Th2 immune responses. Additionally, antibody responses against the glycosylphosphatidylinositol (GPI)-anchored protein merozoite surface protein 1 (MSP-1) were found to be significantly lower in CD1-/- mice compared to wild-type animals. P. berghei-infected MHC class II (MHCII)-/- mice also generated antibodies against MSP-1, suggesting that antibody production against GPI-anchored antigens in response to malaria infection can arisefrom both MHCII-dependent and independent pathways.

Simultaneous neuroprotection and blockade of inflammation reverses autoimmune encephalomyelitis

In multiple sclerosis, the immune system attacks the white matter of the brain and spinal cord, leading to disability and/or paralysis. Myelin, oligodendrocytes and neurons are lost due to the release by immune cells of cytotoxic cytokines, autoantibodies and toxic amounts of the excitatory neurotransmitter glutamate. Experimental autoimmune encephalomyelitis (EAE) is an animal model that exhibits the clinical and pathological features of multiple sclerosis. Current therapies that suppress either the inflammation or glutamate excitotoxicity are partially effective when administered at an early stage of EAE, but cannot block advanced disease. In a multi-faceted approach to combat EAE, we blocked inflammation with an anti-MAdCAM-1 (mucosal addressin cell adhesion molecule-1) monoclonal antibody and simultaneously protected oligodendrocytes and neurons against glutamate-mediated damage with the -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate antagonist 2,3-dihydroxy-6-nitro-7- sulfamoylbenzo(f)quinoxaline (NBQX) and the neuroprotector glycine–proline–glutamic acid (GPE; N-terminal tripeptide of insulin-like growth factor). Remarkably, administration at an advanced stage of unremitting EAE of either a combination of NBQX and GPE, or preferably all three latter reagents, resulted in amelioration of disease and repair of the CNS, as assessed by increased oligodendrocyte survival and remyelination, and corresponding decreased paralysis, inflammation, CNS apoptosis and axonal damage. Each treatment reduced the expression of nitric oxide and a large panel of proinflammatory and immunoregulatory cytokines, in particular IL-6 which plays a critical role in mediating EAE. Mice displayed discernible improvements in all physical features examined. Disease was suppressed for 5 weeks, but relapsed when treatment was suspended, suggesting treatment must be maintained to be effective. The above approaches, which allow CNS repair by inhibiting inflammation and/or simultaneously protect neurons and oligodendrocytes from damage, could thus be effective therapies for multiple sclerosis.

The effects of testosterone on immune function in quail selected for divergent plasma corticosterone response

The immunocompetence handicap hypothesis (ICHH) suggests that the male sex hormone testosterone has a dual effect; it controls the development and expression of male sexually selected signals, and it suppresses the immune system. Therefore only high quality males are able to fully express secondary sexual traits because only they can tolerate the immunosuppressive qualities of testosterone. A modified version of the ICHH suggests that testosterone causes immunosuppression indirectly by increasing the stress hormone corticosterone (CORT). Lines of Japanese quail (Coturnix japonica) selected for divergent responses in levels of plasma CORT were used to test these hypotheses. Within each CORT response line (as well as in a control stock) we manipulated levels of testosterone in castrated quail by treatment with zero (sham), low or high testosterone implants, before testing the birdsʼ humoral immunity and phytohaemagglutinin (PHA)-induced immune response, as well as body condition. The PHA-induced response was not significantly affected by CORT selected line, testosterone treatment or their interaction. There was, however, a significant effect of CORT line on humoral immunity in that the control birds exhibited the greatest antibody production, but there was no significant effect of testosterone manipulation on humoral immunity. The males in the sham implant treatment group had significantly greater mass than the males in the high testosterone group, suggesting a negative effect of high testosterone on general body condition. We discuss these results in the context of current hypotheses in the field of sexual selection.

Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles : implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens

Background

Grass pollen allergens are the most important cause of hay fever and allergic asthma during summer in cool temperate climates. Pollen counts provide a guide to hay fever sufferers. However, grass pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet, grass pollen allergens are known to be associated with atmospheric respirable particles.
Objective

We aimed (1) to determine the concentration of group 5 major allergens in (a) pollen grains of clinically important grass species and (b) atmospheric particles (respirable and nonrespirable) and (2) to compare the atmospheric allergen load with clinical data to assess different risk factors for asthma and hay fever.
Methods

We have performed a continuous 24 h sampling of atmospheric particles greater and lower than 7.2 μm in diameter during the grass pollen season of 1996 and 1997 (17 October 1996–16 January 1997) by means of a high volume cascade impactor at a height of about 15 m above ground in Melbourne. Using Western analysis, we assessed the reactivity of major timothy grass allergen Phl p 5 specific monoclonal antibody (MoAb) against selected pollen extracts. A MoAb-based ELISA was then employed to quantify Phl p 5 and cross-reactive allergens in pollen extracts and atmospheric particles larger and smaller than 7.2 μm.
Results

Phl p 5-specific MoAb detected group 5 allergens in tested grass pollen extracts, indicating that the ELISA employed here determines total group 5 allergen concentrations. On average, 0.05 ng of group 5 allergens were detectable per grass pollen grain. Atmospheric group 5 allergen concentrations in particles > 7.2 μm were significantly correlated with grass pollen counts (rs = 0.842, P < 0.001). On dry days, 37% of the total group 5 allergen load, whereas upon rainfall, 57% of the total load was detected in respirable particles. After rainfall, the number of starch granule equivalents increased up to 10-fold; starch granule equivalent is defined as a hypothetical potential number of airborne starch granules based on known pollen count data. This indicates that rainfall tended to wash out large particles and contributed to an increase in respirable particles containing group 5 allergens by bursting of pollen grains. Four day running means of group 5 allergens in respirable particles and of asthma attendances (delayed by 2 days) were shown to be significantly correlated (P < 0.001).
Conclusion

Here we present, for the first time, an estimation of the total group 5 allergen content in respirable and nonrespirable particles in the atmosphere of Melbourne. These results highlight the different environmental risk factors for hay fever and allergic asthma in patients, as on days of rainfall following high grass pollen count, the risk for asthma sufferers is far greater than on days of high pollen count with no associated rainfall. Moreover, rainfall may also contribute to the release of allergens from fungal spores and, along with the release of free allergen molecules from pollen grains, may be able to interact with other particles such as pollutants (i.e. diesel exhaust carbon particles) to trigger allergic asthma.

Immunologic significance of respirable atmospheric starch granules containing major birch allergen Bet v 1

Background: Birch-pollen allergens are an important cause of early spring hay fever and allergic asthma. Recently, we reported a mechanism for the release of respirable allergenic particles from birch pollen containing the major allergen Bet v 1. In this study, we aimed to assess the immunologic significance of the released Bet v 1-containing starch granules in the environment.

Methods: A two-site monoclonal antibody-based assay (ELISA) was employed to quantitate Bet v 1 in high-volume air sampler filter extracts, and immunogold-labelling was used on sections of these extracts to localize Bet v 1. Immunoblot analyses were performed with pooled sera from patients sensitive to birch pollen.

Results: Atmospheric starch granules contained Bet v 1, and the concentration increased upon light rainfall. Sera from patients allergic to birch allergens recognized extracts from isolated starch granules.

Conclusions: The clinical implications of these findings are that starch granules released from birch pollen are potentially able to trigger allergic asthmatic reactions to Bet v 1, since the allergen occurs in respirable particles. Thus, clinicians can advise asthma patients to remain indoors on days of light rainfall during the birch-pollen season to avoid high levels of allergen exposure.

Major grass pollen allergen Lol p 1 binds to diesel exhaust particles : implications for asthma and air pollution

Background Grass pollen allergens are known to be present in the atmosphere in a range of particle sizes from whole pollen grains (approx. 20 to 55 μim in diameter) to smaller size fractions < 2.5 μ (fine particles, PM2.5). These latter particles are within the respirable range and include allergen-containing starch granules released from within the grains into the atmosphere when grass pollen ruptures in rainfall and are associated with epidemics of thunderstorm asthma during the grass pollen season. The question arises whether grass pollen allergens can interact with other sources of fine particles, particularly those present during episodes of air pollution.

Objective We propose the hypothesis that free grass pollen allergen molecules, derived from dead or burst grains and dispersed in microdroplets of water in aerosols, can bind to fine particles in polluted air.

Methods We used diesel exhaust carbon particles (DECP) derived from the exhaust of a stationary diesel engine, natural highly purified Lol p 1, immunogold labelling with specific monoclonal antibodies and a high voltage transmission electron -microscopic imaging technique

Results DECP are visualized as small carbon spheres, each 30–60 nm in diameter, forming fractal aggregates about 1–2μ in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p I. binds to one defined class of fine particles, DECP.

Conclusion DECP are in the respirable size range, can bind to the major grass pollen allergen Lol p I under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.

Bevacizumab for the treatment of high-grade glioma

Introduction: Gliomas are highly vascular and rich in vascular endothelial growth factor (VEGF) that promotes angiogenesis. Bevacizumab is a monoclonal antibody against VEGF inhibiting angiogenesis by preventing receptor activation. Phase II clinical trials using bevacizumab in both newly diagnosed and recurrent high-grade glioma (HGG) showed promising results.

Areas covered: This is a review of clinical trials investigating bevacizumab in newly diagnosed and recurrent HGGs with a focus on outcome results. A future perspective about the expected role of bevacizumab is given. Bevacizumab efficacy, safety and tolerability, the combination of radiation and bevacizumab as well as the use of bevacizumab to treat pseudoprogression are discussed. Further criteria of response evaluation needed to be adjusted in the age of anti-angiogenic therapy and this will be discussed.

Expert opinion: Bevacizumab has been shown to be safe and tolerable in HGG. In the recurrent disease setting, bevacizumab alone might be sufficient for a clinical benefit and is currently approved as a single agent for this indication. While clinical trials demonstrate a prolonged progression-free survival in bevacizumab-treated HGG, a benefit on OS has not been demonstrated yet. Bevacizumab has also been introduced into other settings in neuro-oncology including concurrent administration with re-irradiation for recurrent HGG.

Intracranial hemorrhage (ICH) in cancer patients treated with bevacizumab : the Memorial Sloan-Kettering experience

Background: Bevacizumab is a monoclonal antibody targeting vascular endothelial growth factor approved for recurrent glioblastoma (GBM), metastatic breast, colorectal and non-small-cell lung cancers (NSCLC). There has been a potentially increased risk of intracranial hemorrhage (ICH) in patients receiving bevacizumab.

Methods: We retrospectively identified patients with ICH who received bevacizumab between 1 January 2001 and 10 January 2009.

Results: We identified 1024 patients with ICH, 4191 patients who received bevacizumab and 12 (0.3%) who met both our criteria. There were eight women and four men with a median age of 66 years. Primary cancers were ovarian (n = 3), NSCLC (n = 3), colon (n = 1), angiosarcoma (n = 1) and GBM (n = 4). Intracranial tumors were present in 9 of the 12 patients; the remaining three (25%) had no evidence of intracranial pathology. Two hundred and fifty-seven patients with these same primary pathologies and brain tumors were treated with bevacizumab; ICH was seen in nine (3.7%), which was comparable to the 3.6% frequency seen in comparable patients not receiving bevacizumab.

Conclusions: ICH with bevacizumab treatment in this population is rare and does not appear to increase its frequency over the baseline rate of ICH in a comparable population. Most bevacizumab-related ICH occurs into central nervous system tumors but spontaneous hemorrhages were seen.

The stress and metabolic responses of juvenile Atlantic cod Gadus morhua L. to an acute thermal challenge

Survival, oxygen consumption (M_O2), total plasma cortisol and glucose levels and gill heat-shock protein 70 (hsp70) expression were measured in 10 and 50 g juvenile Atlantic cod Gadus morhua during an acute temperature increase (2° C h⁻¹) to their critical thermal maximum. Ninety three per cent of the fish in both size classes survived to 24° C; however, mortality was 100% within 15 min of reaching this temperature. The M_O2 for both size classes increased significantly with temperature, reaching peak values at 22° C that were c. 2·8-fold those of control (10° C) fish. Resting plasma cortisol and glucose levels were lower in 10 g as compared to 50 g fish. Plasma glucose levels were highly variable in both size classes, and significant increases were only seen at >22° C for the 10 g fish. In contrast, plasma cortisol showed an exponential increase with temperature starting at 16° C in both size classes, and reached maximum levels at 22° C that were 19-fold (10 g fish) and 35-fold (50 g fish) higher than their respective control groups. Both the constitutive (73 kDa) and inducible (72 kDa) isoforms of hsp70 were detected in both size classes using the widely utilized mouse monoclonal antibody. Expression of these isoforms, however, did not change when Atlantic cod were exposed to elevated temperature, and the 72 kDa isoform was not detected using salmonid-specific antibodies. These results indicate that juvenile Atlantic cod are very sensitive to acute increases in water temperature. In addition, they (1) show that M_O2and plasma cortisol, but not plasma glucose or gill hsp 70 levels, are sensitive indicators of thermal stress in Atlantic cod and (2) support previous reports that the upper critical temperature for this species is 16° C.

Primary systemic therapy in HER2-amplified breast cancer : a clinical review

Primary systemic therapy (PST) in early breast cancer is utilized in locally advanced breast tumors and when breast-conserving surgery is desirable. In addition, the PST setting provides an opportunity to monitor response including histopathological and biomarker examination of the tumor and host tissues before and after systemic therapy. Trastuzumab is a monoclonal antibody targeting the hEGF receptor that is overexpressed in 15–20% of breast tumors. Trastuzumab is effective in prolonging survival when used to treat women with hEGF receptor overexpressed tumors, both in adjuvant and metastatic disease settings. Trastuzumab has also shown promising activity in PST/neoadjuvant studies by achieving high rates of complete pathologic response. This is a review of clinical studies that incorporated trastuzumab in PST and/or neoadjuvant chemotherapy, including the results of recently reported studies using trastuzumab in combination with other novel therapies such as lapatinib or pertuzumab.