In vitro proliferation of saffron (Crocus sativus L.) stigma

Excised young intact stigmas plus ovaries of Crocus sativus L. were cultured on Linsmaier-Skoog media supplemented with either a cytokinin or an auxin alone or in combinations. Benzyladenine and kinetin at concentrations of 0.1, 1, and 5 mgl^-1 supported growth, and crocin was biosynthesized in the stigmas in vitro. Auxins had little effect. Young excised single stigmas or half ovaries were also cultured so as to form stigma-like structures in order to explore a possible new approach to industrial production of the spice, saffron. On Linsmaier-Skoog and Nitsch media supplemented with kinetin at concentration of 1 or 5 mgl^-1 and alpha-naphthalene acetic acid or indole-butyric acid at concentration of 0.1 or 10 mgl^-1 in combinations, stigma-like structures appeared directly and indirectly (through meristematic tissue), grew and matured. The maximum number of structures were 75 per half ovary. Three kinds of yellow pigments including crocin were tentatively identified by TLC in the stigma-like structures as was the case for the in vivo grown natural stigma, although the contents were lower.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-acetic acid - IBA indole-butyric acid - NAA alpha-naphthalene-acetic acid - TLC thin layer chromatography     

Monoclonal antibodies to plant growth regulators. II. Indole-3-acetic acid

The production and characterization of high-affinity monoclonal antibodies suitable for the radio- and enzymeimmunoassay of the endogenous plant growth regulator, indole-3-acetic acid (IAA), is reported. Hybridomas were produced by fusion of NS 1 myeloma cells with spleen cells from Balb/c mice immunized with IAA-bovine serum albumin conjugates. From an initial collection of 158 wells containing cells secreting monoclonal antibodies against IAA, seven were used to derive cell clones. Three of these are described here. They secrete immunoglobulin (IgG2a or IgG2b) of high affinity and specificity for IAA methyl ester and can be used to quantite picogram amounts of this compound in plant extracts by radio- and enzymeimmunoassay.     

A hyman hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by Pseudomonas aeruginosa exotoxin A

By fusing a human hybridoma producing an IgG2 antibody against human A431 epidermoid carcinoma cells with an Epstein-Barr virus-transformed human B lymphocyte producing an IgG2 antibody against Pseudomonas aeruginosa exotoxin A, we established a hybrid hybridoma producing a bispecific monoclonal antibody reacting with both A431 cells and the exotoxin. Human IgG was purified from the culture supernatant of the hybrid hybridoma, and the bispecific monoclonal antibody in the IgG preparation was further separated from the two parental antibodies by hydroxyapatite high-performance liquid chromatography. The human bispecific monoclonal antibody thus obtained efficiently targeted the antibody-reative cells, A431, for attack by the exotoxin in vitro.Abbreviations bs mAb Bispecific Monoclonal Antibody - HRP Horseradish Peroxidase - MHA Mixed Hemadsorption Assay - MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide - PEA Pseudomonas aeruginosa Exotoxin A - PEG Polyethylene Glycol     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

Summary The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumourspecific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with¹²⁵iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b > IgG2a > IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab)₂ preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.     

Humoral and cellular responses of colorectal cancer patients treated with monoclonal antibodies and interferon γ

Summary Fifteen patients with metastatic gastrointestinal adenocarcinomas were treated with low doses of recombinant human interferon (rh-IFN) and a mixture of monoclonal antibodies (mAb) that bind to tumor cells. All antibodies were of the IgG2a isotype and interact with human effector cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). Natural killer lysis against K562 cells by peripheral blood mononuclear cells purified from patients' blood was enhanced in all patients at day 3 during IFN treatment. Monocytes from two patients had increased ADCC levels. Increase in the percentage of monocytes able to bind mouse IgG2a was detected by Fc receptor flow cytometry analysis 24 h after the first IFN infusion. However, 3 days later, the percentage of fluorescent cells had fallen below baseline levels. The analysis of patients' sera showed that at day 2 after mAb infusion, only 50% of the circulating mouse IgG was immunoreactive, and after 1 week, only traces of immunoreactive mouse IgG were detected. All patients developed a human anti-(mouse Ig) response of IgG, IgM and IgA isotypes, although only low levels of anti-idiotypic antibodies were detected at the time of testing (up to 9 weeks) after mAb infusion. No difference in the IgG subclasses of anti-(mouse Ig) antibody was observed between patients treated with mAb and IFN and patients treated with mAb alone.This work was supported by a fellowship from the Minister of Foreign Affairs, France, and National Institutes of Health Grants CA10815, CA25874 and CA21124.     

Mechanisms of stable serum resistance of Neisseria gonorrhoeae

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent sera from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by immunoglobulin G (IgG) or F(ab)₂ isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, contained most of the blocking activity in IgG. In addition, immune convalescent DGI serum, which did not exhibit bactericidal activity, was restored to killing by selective immunodepletion of protein III antibodies. Blocking IgG or F(ab)₂ prepared from IgG, partially inhibited binding of bactericidal antibody to N. gonorrhoeae. Also, binding of a monoclonal antibody recognizing N. gonorrhoeae outer membrane protein PIII was almost completely inhibited by blocking F(ab)₂.Presensitization of N. gonorrhoeae with increasing concentrations of blocking IgG or F(ab)₂ before incubation with bactericidal antibody and an antibody free source of complement, increased consumption and deposition of the third component of human complement (C3) and the ninth component of complement (C9) but inhibited killing in dose-related fashion.     

IgG4-related disease: why high IgG4 and fibrosis?

The hallmarks of IgG4-related disease (IgG4-RD) are lymphoplasmacytic tissue infiltration with a predominance of IgG4-positive plasma cells, accompanied by fibrosis, obliterative phlebitis, dacryoadenitis, and elevated levels of IgG4. In a recent issue of Arthritis Research & Therapy, Tsuboi and colleagues demonstrated that regulatory T (Treg) cell-and T helper 2 (Th2) cell-derived cytokines contribute to the pathogenesis of Mikulicz''s disease, an activation pathway that appears to be common for IgG4-RD. Additional organ-specific factors may account for the different organ involvement of IgG4-RD.IgG4-related disease (IgG4-RD) is a newly categorized disease entity initially recognized in Japan but increasingly also in other parts of the world [1,2]. Most often the diagnosis is made in patients with autoimmune pancreatitis. Additional presentations include patients with lacrimal and salivary gland involvement, formerly Mikulicz''s disease (MD), which was once thought to be a subset of Sjögren''s syndrome (SS).The hallmarks of IgG4-RD are lymphoplasmacytic tissue infiltration with a dominance of IgG4-positive plasma cells, accompanied by fibrosis, obliterative phlebitis, dacryoadenitis, and elevated levels of IgG4. The pathogenesis of IgG4-RD is poorly understood; findings consistent with both an autoimmune disorder and an allergic disorder are present [1,2].IgG4 production is controlled primarily by T helper 2 (Th2) cells [3,4]. Th2 cytokines interleukin-4 (IL-4) and IL-13 enhance the production of IgG4 and IgE. In contrast, IL-10, IL-12, and IL-21 shift the balance between IgG4 and IgE, favoring IgG4. In the Th2 cytokine-driven immune reaction, IgG4 production is induced preferentially by the activation of IL-10 produced by regulatory T (Treg) cells [3]. Thus, selective IgG4 induction is referred to as the combined effect of Th2 and Treg cells.In a recent issue of Arthritis Research & Therapy, Tsuboi and colleagues [5] analyzed the expression of IgG4-specific class switch molecules such as Th2 cytokines (IL-4 and IL-13) and Treg cytokines (IL-10 and TGF-β), IgG4-nonspecific B cell regulatory molecules (CD40, CD154, BAFF, APRIL, and IRF4), and activation-induced cytidine deaminase (AID) in the labial salivary glands (LSGs) and peripheral blood mononuclear cells (PBMCs) from patients with IgG4-RD (MD) and SS. The authors provided evidence that IL-10, TGF-β, and AID were overexpressed in LSGs from IgG4-RD (MD) compared with those in patients with SS, suggesting that Treg cytokines (IL-10 and TGF-β) contribute to IgG4-specifc class switch recombination and fibrosis in patients with IgG4-RD (MD) in combination with the IgG4-unrelated molecule, AID (Figure ​(Figure11).Open in a separate windowFigure 1Molecular mechanism of IgG4-related disease. AID, activation-induced cytidine deaminase; IL, interleukin; TGF-β, transforming growth factor-beta; Th, T helper; Treg, regulatory T.Very recently, Tanaka and colleagues [6] examined the Th1, Th2, and Treg cytokine expression in LSGs from patients with IgG4-RD and SS. The authors showed that the levels of mRNA for both Th2 and Treg cytokines were significantly higher in LSGs from patients with IgG4-RD (MD) but that the expressions of Th1 and Th2 cytokines were higher in LSGs from patients with SS. The upregulation of Treg cytokines is identical to the findings reported by Tsuboi and colleagues [5], indicating that Treg cells play an important role in the pathogenesis of IgG4-RD (MD). In contrast, Tsuboi and colleagues showed that Th2 cytokines such as IL-4 and IL-13 were not significantly overexpressed in LSGs from patients with IgG4-RD (MD) but were increased if compared with those in healthy subjects. This finding supports the notion that Th2 cytokines such as IL-4 and IL-13 play a common B cell-activating role in both IgG4-RD (MD) and SS. Contrary to Th2 and Treg cytokines, Th1 cytokines were upregulated only in LSGs from patients with SS [6], suggesting that Th1 cells function as key players in the pathogenesis of SS.Consistent with the findings in MD, analyses of the expression of cytokines in inflammatory lesions from patients with IgG4-related sclerosing pancreatitis and cholangitis [7] or tubulointerstitial nephritis [8] showed that tissue mRNA expression of Th2 (IL-4) and Treg cytokines (IL-10 and TGF-β) was substantially higher than in other diseases. Many mononuclear cells expressing IL-4 or IL-10 were identifiable in affected organs by in situ hybridization [7]. Moreover, circulating CD4⁺CD25⁺Treg cells were significantly increased in PBMCs from patients with autoimmune pancreatitis [9].Further examinations should shed light on the molecular mechanisms controlling the activation of this pathway.     

Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma

Summary Serum IgG fractions from a large and homogenous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the non-selective CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.     

The production of a “universal developer” for the immunological detection of human IgG and its application in immunodiagnostics

Summary This study describes the development of a bispecific monoclonal antibody capable of the simultaneous recognition of horseradish peroxidase (HRP) and human IgG. This antibody, coded McC2, has been applied in a novel manner as a universal developing reagent for the detection of human IgG. McC2 cross-reacts with all human IgG subtypes and was found to recognise an epitope on the Fe portion of human IgG. McC2 does not cross-react with human IgM or IgA. This bi-specific antibody belongs to the mouse IgG₁ subclass. McC2 was used for the detection of human IgG in a simple one step enzyme-linked immunosorbant assay (ELISA). Use of this bi-specific antibody in this assay resulted in an excellent signal to noise ratio with background in negative control wells virtually nonexistent. McC2 was also applied in a clinical diagnostic test for the detection of auto anti-nuclear antibodies in patient sera. McC2 was substituted, in a blind study, for a HRP-conjugated second antibody supplied with the test kit. All sera were tested both with the kit's second antibody and McC2. When using McC2, we obtained no false positive results whereas five false positives were obtained when using the kit's second antibody. However, one false negative result was obtained with the use of McC2 as a developing reagent while none were noted with the use of the kit's second antibody.This study demonstrates the potential use of bi-specific universal developers in a wide variety of immunobased techniques as well as the potential advantages for the production of a complete panel of bi-specific developing monoclonal antibodies against IgGs from a number of different species.     

Regulation of IgE activity in inhalational tolerance via formation of IgG anti-IgE/IgE immune complexes

Background

Allergic asthma is an inflammatory disorder of the airways that results from inappropriate production of IgE against harmless, environmental antigens. Sequestration of free IgE using humanized IgG anti-IgE is an effective therapy for asthma and other atopic disorders. However, the status of free IgE in subjects who have naturally developed immune tolerance to inhaled antigens has not been well studied.

Methods

C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) for 7 days to induce allergic airway disease (AAD) or 6 weeks to induce a state of local inhalational tolerance (LIT). Serum from AAD or LIT mice, diluted to achieve equivalent levels of total OVA-specific IgE, was used to sensitize rat basophil leukemia cells for allergen-mediated degranulation. Levels of degranulation were measured in relation to serum concentrations of free IgE and IgG anti-IgE/IgE immune complexes.

Results

Serum from AAD animals induced a greater degree of basophil degranulation than serum from LIT animals. These results correlated with higher levels of free IgE in AAD animals, whereas LIT mice demonstrated a significant increase in IgG anti-IgE/IgE immune complexes relative to their diseased counterparts.

Conclusions

Sequestration of free IgE by naturally occurring IgG anti-IgE may aid in the development of immune tolerance against inhaled allergens. The decrease in bioavailability of free IgE may, in turn, contribute to the overall reduction of asthma symptoms via a mechanism that mimics the therapeutic effects of humanized IgG anti-IgE.

     

Anti-Human Herpesvirus 6A/B IgG Correlates with Relapses and Progression in Multiple Sclerosis

Objective

To analyze the titers of the IgG and IgM antibodies against human herpesvirus 6A/B (HHV-6A/B) in multiple sclerosis (MS) patients treated with different disease modified therapies (DMTs) along two-years of follow-up.

Methods

We collected 2163 serum samples from 596 MS; for 301 MS patients a 2-years follow-up was performed. Serum samples of 337 healthy controls were also analyzed. Anti-HHV-6A/B IgG and IgM were analyzed by ELISA (Panbio).

Results

We found that 129/187 (69.0%) MS patients with a decrease of the anti-HHV-6A/B IgG titers after 2-years with DMTs were free of relapses and progression vs. 46/113 (40.7%) of MS patients with an increase of the anti-HHV-6A/B IgG titers (p = 0.0000015); the higher significance was found for natalizumab. Furthermore, we found that anti-HHV-6A/B IgG titers reached their highest value two weeks before the relapse (p = 0.0142), while the anti-HHV-6A/B IgM titers reached their highest value one month before the relapse (p = 0.0344).

Conclusion

The measurement of the anti-HHV-6A/B IgG titers could be a good biomarker of clinical response to the different DMTs. The increase of the anti-HHV-6A/B IgG and IgM titers predicts the upcoming clinical relapses. However, further longitudinal studies are needed to validate these results.     

Effect of Total Suspended Particulate Matter in the Air on Inflammation Factors and Apoptotic Markers in Diabetic Rats: The Protective Effect of Insulin and Crocin

Background:The effect of total suspended particulate matter (TSP) was investigated on the expression of inflammatory and apoptotic factors in diabetic rats, and the effect of crocin and insulin was examined on these factors.Methods:Fifty-four adult male wistar rats were divided into nine experimental groups: control group, crocin group (received crocin, 50 mg/kg), diabetic group (received a single dose of alloxan at 120 mg/kg, IP), TSP group (5 mg/kg TSP instilled intratracheally), diabetic-crocin group (received crocin at 50 mg/kg after the induction of diabetes by alloxan (120 mg/kg)), diabetic-insulin group (received regular insulin (5 U/kg), crocin-TSP group (received crocin at 50 mg/kg, IP, and then 5 mg/kg TSP was instilled intratracheally), diabetic-TSP-insulin group (after receiving alloxan (120 mg/kg) and instilling TSP (5 mg/kg, intratracheally), a single dose (5 U/kg) of regular insulin), and diabetic-TSP-crocin group (after receiving alloxan (120 mg/kg) and instilling TSP (5 mg/kg, intratracheally), a single dose of crocin (50 mg/kg, IP)). Quantitative real-time PCR was performed to measure the expression of the mRNAs of apoptotic (Bax and Bcl2) and inflammatory mediators (TNFα, COX2, iNOS/eNOS) in Wistar rats.Results:In diabetic and TSP groups the inflammatory factors and BAX/Bcl2 ratio significantly increased compared to the control group. In diabetic-TSP-insulin and diabetic-TSP-crocin, a significant decrease was observed in the rate of inflammatory factors and BAX/Bcl2 ratio.Conclusion:The results suggested that diabetes and exposure to TSP increase the rate of apoptosis and inflammation, and also demonstrated the anti-apoptotic and anti-inflammation role of insulin and crocin.Key Words: Apoptosis, Crocin, Diabetes, Inflammation, Insulin, TSP     

Immunoglobulin classes of human serum antibodies in vaginal candidiasis

The titre and immunoglobulin class of antibodies against Candida albicans in serum from 60 non-pregnant women was determined. IgG titres up to 132, IgA titres up to 18, and IgM titres up to 14 were detected in 30 women with vaginal candidiasis. Similar titres were found in 20 women harbouring yeasts in the mouth or rectum, and in 10 women who were not harbouring yeasts in the vagina, mouth or rectum. Serum fractionation confirmed that antibodies to C. albicans are found in the three immunoglobulin classes and that these antibodies reside in highest titre in the IgG class. No secretory IgA antibodies against C. albicans were detected in the serum of these women.     

Crocin Abrogates Carbon Tetrachloride‐Induced Renal Toxicity in Rats via Modulation of Metabolizing Enzymes and Diminution of Oxidative Stress,Apoptosis, and Inflammatory Cytokines

This work aimed at investigating the potential modulatory effects and mechanisms of crocin against CCl₄‐induced nephrotoxicity. Forty male rats were allocated for three weeks treatment with corn oil, CCl₄, crocin, or crocin plus CCl₄. Crocin effectively mitigated CCl₄‐induced kidney injury as evidenced by amelioration of alterations in kidney histopathology, renal weight/100 g body weight ratio and kidney functions. Crocin modulated CCl₄‐induced disturbance of kidney cytochrom‐P450 subfamily 2E1 and glutathione‐S‐transferase. The attenuation of crocin to kidney injury was also associated with suppression of oxidative stress via reduction of lipid peroxides along with induction of renal glutathione content and enhancement of superoxide dismutase, glutathione peroxidase, and catalase activities. Crocin mitigated CCl₄‐induced elevation of the renal levels of tumor necrosis factor‐alpha, interleukin‐6, prostaglandin E2, and active caspases‐3. Collectively, crocin alleviated CCl₄‐induced renal damage via modulation of kidney metabolizing enzymes, suppression of oxidative stress, inhibition of inflammatory cytokines, PGE2, and active caspase3 in kidney.     

Effect of Antenatal Parasitic Infections on Anti-vaccine IgG Levels in Children: A Prospective Birth Cohort Study in Kenya

Background

Parasitic infections are prevalent among pregnant women in sub-Saharan Africa. We investigated whether prenatal exposure to malaria and/or helminths affects the pattern of infant immune responses to standard vaccinations against Haemophilus influenzae (Hib), diphtheria (DT), hepatitis B (Hep B) and tetanus toxoid (TT).

Methods and Findings

450 Kenyan women were tested for malaria, schistosomiasis, lymphatic filariasis (LF), and intestinal helminths during pregnancy. After three standard vaccinations at 6, 10 and 14 weeks, their newborns were followed biannually to age 36 months and tested for absolute levels of IgG against Hib, DT, Hep B, and TT at each time point. Newborns’ cord blood (CB) lymphocyte responses to malaria blood-stage antigens, soluble Schistosoma haematobium worm antigen (SWAP), and filaria antigen (BMA) were also assessed. Three immunophenotype categories were compared: i) tolerant (those having Plasmodium-, Schistosoma-, or Wuchereria-infected mothers but lacking respective Th1/Th2-type recall responses at birth to malaria antigens, SWAP, or BMA); ii) sensitized (those with infected/uninfected mothers and detectable Th1/Th2-type CB recall response to respective parasite antigen); or iii) unexposed (no evidence of maternal infection or CB recall response).Overall, 78.9% of mothers were infected with LF (44.7%), schistosomiasis (32.4%), malaria (27.6%) or hookworm (33.8%). Antenatal maternal malaria, LF, and hookworm were independently associated with significantly lower Hib-specific IgG. Presence of multiple maternal infections was associated with lower infant IgG levels against Hib and DT antigens post-vaccination. Post-vaccination IgG levels were also significantly associated with immunophenotype: malaria-tolerized infants had reduced response to DT, whereas filaria-tolerized infants showed reduced response to Hib.

Conclusions

There is an impaired ability to develop IgG antibody responses to key protective antigens of Hib and diphtheria in infants of mothers infected with malaria and/or helminths during pregnancy. These findings highlight the importance of control and prevention of parasitic infections among pregnant women.     

Mechanisms of the Effects of Crocin on Aggregation and Deposition of Aβ1–40 Fibrils in Alzheimer’s Disease

Alzheimer??s disease (AD) is among the most important health-care problems in the world. The two pathological hallmarks of AD are extracellular neuritic amyloid plaques and intracellular neurofibrillary tangles. The aggregation of A?? and ??-sheet formation are considered to be the critical events which render these peptides neurotoxic. AD is affecting a large percentage of the elderly around the world. Many studies have been done on drugs to cure or at least slow Alzheimer??s disease. Most drugs produced for this disease aim at compensating for the performance of specific cell groups affected by the disease or restoring the function of these cells.This study examined the interaction of crocin, the main pigment of saffron, with the amyloid-?? peptides 1?+?40 (A?? 40) to determine the effects on peptide conformation and fibril formation using fluorescence spectroscopy, CD spectroscopy and electron microscopy. ThT data demonstrated the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of A??40. Incubation of pre-formed A??40 fibrils with crocin resulted in extensive lateral aggregation and precipitation of the fibrils. Consistent with this, electron microscopy data indicated that crocin decreased the number of fibrils formed and significantly reduced the average fibril length of A??40 as assessed by low levels of thioflavin T binding data. The mechanism by which, crocin prevented fibril formation was demonstrated by ANS binding assay and CD spectroscopy. In summary, crocin interacts with A?? peptides and prevents amyloid formation. This means that it has the potential to be an important therapeutic drug against AD.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice

The cytotoxic antibody response to the H-2D^b alloantigen has been investigated in ten strains of the C57BL/10 background. Three types of responses could be distinguished: no detectable response, an IgM response, and an IgG response. The IgG response is influenced by the D and probably the I-A region of the H-2 complex, whereas the IgM response is dependent on the allele for the E chain. The hypothesis is proposed that regulatory T cells, which recognize the antigen in context of self MHC molecules, determine the outcome of an anti-H-2D^b immunization in which the I-E molecule restricts the IgM response and the I-A molecule restricts the IgG response; the D molecule is probably responsible for activation of suppressor T cells which suppress only the IgG response.     

Functional autoantibodies against SSX-2 and NY-ESO-1 in multiple myeloma patients after allogeneic stem cell transplantation

Background

Multiple myeloma (MM) is the malignancy with the most frequent expression of the highly immunogenic cancer–testis antigens (CTA), and we have performed the first analysis of longitudinal expression, immunological properties, and fine specificity of CTA-specific antibody responses in MM.

Methods

Frequency and characteristics of antibody responses against cancer–testis antigens MAGE-A3, NY-ESO-1, PRAME, and SSX-2 were analyzed using peripheral blood (N = 1094) and bone marrow (N = 200) plasma samples from 194 MM patients.

Results

We found that antibody responses against CTA were surprisingly rare, only 2.6 and 3.1 % of patients evidenced NY-ESO-1- and SSX-2-specific antibodies, respectively. NY-ESO-1-specific responses were observed during disease progression, while anti-SSX-2 antibodies appeared after allogeneic stem cell transplantation and persisted during clinical remission. We found that NY-ESO-1- and SSX-2-specific antibodies were both capable of activating complement and increasing CTA uptake by antigen-presenting cells. SSX-2-specific antibodies were restricted to IgG3, NY-ESO-1 responses to IgG1 and IgG3. Remarkably, NY-ESO-1-positive sera recognized various non-contiguous regions, while SSX-2-specific responses were directed against a single 6mer epitope, SSX-2^85–90.

Conclusions

We conclude that primary autoantibodies against intracellular MM-specific tumor antigens SSX-2 and NY-ESO-1 are rare but functional. While their contribution to disease control still remains unclear, our data demonstrate their theoretic ability to affect cellular anti-tumor immunity by formation and uptake of mono- and polyvalent immune complexes.

     

The effect of crocin and safranal, constituents of saffron, against subacute effect of diazinon on hematological and genotoxicity indices in rats

In this study, the effect of crocin and safranal was studied against subacute toxicity of diazinon (DZN) on hematological and genotoxicity indices in rats. The rats were divided into 16 groups consisted of 6 rats in control, diazinon, vitamin E, vitamin E and DZN, crocin (3 doses), crocin (3 doses) and DZN, safranal (3 doses), safranal (3 doses) and DZN groups. Vitamin E (200 IU/kg), safranal at doses 0.025, 0.05 and 0.1 ml/kg and crocin at doses 50, 100 and 200 mg/kg were injected intraperitoneally to rats three times per week alone or with DZN (20 mg/kg/day, orally) for 4 weeks. Hematological parameters were evaluated at the end of 4 weeks. The evaluation of genotoxicity was done using the micronucleus assay. Vitamin E and, at lower doses, safranal (0.025 and 0.05 ml/kg) and crocin (50 mg/kg) restored the reduction of red blood cell, hemoglobin and hematocrit indices induced by DZN. These agents at some doses also prevented the reduction in platelets counts indices in diazinon treated group. A significant increase in reticulocyte was induced by diazinon. Vitamin E, safranal (0.025 or 0.05 ml/kg) and all doses of crocin decreased this effect of diazinon. In all doses vitamin E, crocin and safranal did not inhibit the effect of diazinon on RBC cholinesterase activity. A significant increase in micronucleus indices was seen with diazinon. Vitamin E, safranal and crocin could not prevent this genotoxicity. This study showed that vitamin E, safranal and crocin (without effects on cholinesterase) reduced diazinon hematological toxicity, but they did not prevent the genotoxicity induced by diazinon.