Efficacy, acceptability, and tolerability of all available treatments for insomnia in the elderly: a systematic review and network meta-analysis.

OBJECTIVES: Symptoms of insomnia are highly prevalent in the elderly. A significant number of pharmacological and non-pharmacological interventions exist, but, up-to-date, their comparative efficacy and safety has not been sufficiently assessed. METHODS: We integrated the randomized evidence from every available treatment for insomnia in the elderly (>65 years) by performing a network meta-analysis. Several electronic databases were searched up to May 25, 2019. The two primary outcomes were total sleep time and sleep quality. Data for other 6 efficacy and 8 safety outcomes were also analyzed. RESULTS: Fifty-three RCTs with 6832 participants (75 years old on average) were included, 43 of which examined the efficacy of one or more drugs. Ten RCTs examined the efficacy of non-pharmacological interventions and were evaluated only with pairwise meta-analyses because they were disconnected from the network. The overall confidence in the evidence was very low primarily due to the small amount of data per comparison and their sparse connectedness. Several benzodiazepines, antidepressants, and z-drugs performed better in both primary outcomes, but few comparisons had data from more than one trial. The limited evidence on non-pharmacological interventions suggested that acupressure, auricular acupuncture, mindfulness-based stress reduction program, and tart cherry juice were better than their control interventions. Regarding safety, no clear differences were detected among interventions due to large uncertainty. CONCLUSIONS: Insufficient evidence exists on which intervention is more efficacious for elderly patients with insomnia. More RCTs, with longer duration, making more direct interventions among active treatments and presenting more outcomes are urgently needed.

Microtubule-associated protein tau facilitates the targeted killing of proliferating cancer cells in vitro and in a xenograft mouse tumour model in vivo.

BACKGROUND: Antibody drug conjugates (ADCs) and immunotoxins (ITs) are promising anticancer immunotherapeutics. Despite their encouraging performance in clinical trials, both ADCs and ITs often suffer from disadvantages such as stoichiometrically undefined chemical linkage of the cytotoxic payload (ADCs) and the potential immunogenicity of toxins derived from bacteria and plants (ITs). METHODS: Human microtubule-associated protein tau (MAP) was cloned in-frame with human EGF, expressed in E. coli and purified by standard chromatographic methods. The in vitro activity was confirmed by flow cytometry, cell viability assays and tubulin polymerisation assay. The in vivo efficacy was demonstrated using noninvasive far-red in vivo imaging. RESULTS: The EGF-MAP selectively induced apoptosis in EGFR-overexpressing proliferating cancer cells through stabilisation of microtubules. Nonproliferating cells were not affected, demonstrating superior selectivity of EGF-MAP for cancer cells. The EGF-MAP was well tolerated at high doses in mice compared with the ETA'-based control. The in vivo efficacy of EGF-MAP was demonstrated in a tumour xenograft mouse model. CONCLUSION: Our data indicate the general mechanism of action for a new class of human immunotherapeutic reagents suitable for the treatment of cancer. This approach combines the binding specificity of targeting ligands with the selective cytotoxicity of MAP towards proliferating cells.

Immunology in the clinic review series; focus on type 1 diabetes and viruses: the innate immune response to enteroviruses and its possible role in regulating type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease arising as a consequence of a misdirected T cell response to the pancreatic beta cell. In recent years, there has been a growing interest in the innate immune system as a regulator of disease development. Genome-wide association studies have identified diabetes-associated polymorphisms in genes encoding proteins with functions related to the innate immune response. Moreover, enteroviruses, known to activate a strong innate immune response, have been implicated in the disease pathogenesis. In this review, we discuss the innate immune response elicited by enteroviruses and how this response may regulate T1D development.

Recombinant, ETA'-based CD64 immunotoxins: improved efficacy by increased valency, both in vitro and in vivo in a chronic cutaneous inflammation model in human CD64 transgenic mice.

BACKGROUND: Dysregulated, activated macrophages play a pivotal role in chronic inflammatory diseases such as arthritis and atopic dermatitis. These cells display increased expression of the high-affinity Fcgamma receptor (CD64), making them ideal targets for CD64-specific immunotoxins. We previously showed that a chemically linked immunotoxin, the monoclonal H22-RicinA, specifically eliminated infiltrating activated macrophages and resolved chronic cutaneous inflammation. However, several disadvantages are associated with classic immunotoxins, and we therefore followed a fusion protein strategy to express the antigen-binding site alone (scFv H22) fused to a derivative of Pseudomonas exotoxin A (ETA'). OBJECTIVES: To assess the potential effect of increased valency on efficacy, we produced monovalent [H22(scFv)-ETA'] and bivalent [H22(scFv)(2)-ETA'] versions and evaluated their potential for eliminating activated macrophages both in vitro and in vivo. METHODS: Both immunotoxins were produced by bacterial fermentation. Binding was assessed by flow cytometry on the monocytic CD64+ cell line U937. Toxicity was analysed by XTT and apoptosis induction by annexin V bioassay. The in vivo effect was tested in a human CD64 transgenic mouse model for cutaneous inflammation. RESULTS: The cytotoxic effects of both immunotoxins were clearly due to apoptosis with an IC(50) of 140 pmol L(-1) for monovalent and only 14 pmol L(-1) for the divalent version. In vivo treatment with H22(scFv)-ETA' reduced CD64+ activated macrophages to 21% of their initial numbers whereas H22(scFv)(2)-ETA' treatment reduced these cells to 4.8% (P < 0.001). CONCLUSIONS: These data clearly show increased efficacy due to increased valency of the anti-CD64 immunotoxin. Both recombinant immunotoxins have a low IC(50), making them suitable for the treatment of diseases involving dysregulated, activated macrophages.

A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model.

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) gamma-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

Human angiogenin fused to human CD30 ligand (Ang-CD30L) exhibits specific cytotoxicity against CD30-positive lymphoma.

A number of different immunotoxins composed of cell-specific targeting structures coupled to plant or bacterial toxins have increasingly been evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based fusion toxin (Ang-CD30L) using the human RNase angiogenin. The completely human fusion gene was inserted into a pET-based expression plasmid. Transformed Escherichia coli BL21(DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the M(r) 37,000 His(10)-tagged Ang-CD30L was directed into the periplasmic space and functionally purified by a combination of metal ion affinity followed by enterokinase cleavage of the His(10)-Tag and molecular size chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays showing specific activity against CD30(+) Hodgkin-derived cells. Specific binding activity of Ang-CD30L was verified by competition with anti-CD30 monoclonal antibody Ki-4 and commercially available CD30L-CD8 chimeric protein. Ang-CD30L showed RNase activity in vitro. The human recombinant immunotoxin showed significant toxicity toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and L540Cy) and exhibited highest cytotoxicity against L540 cells (IC(50) = 8 ng/ml) as determined by cell proliferation assays. CD30 specificity was confirmed by competitive toxicity assays. This is the first report on the specific cytotoxicity of a recombinant completely human fusion toxin with possibly largely reduced immunogenicity for the treatment of CD30-positive malignancies.

CD161(int)CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut.

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161(int)CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

Selection of scFv phages on intact cells under low pH conditions leads to a significant loss of insert-free phages.

Display of functional antibody fragments on the surface of filamentous bacteriophages allows fast selection of specific phage antibodies against a variety of target antigens. However, enrichment of single chain variable fragment (scFv)-displaying phages is often hampered by the abundance of bacteriophages lacking antibody fragments. Moderate adhesive binding activities and production advantages of these "empty" phages results in their subsequent enrichment during selection on target cells. To date, very limited effort has been made to develop strategies removing nonspecific binding phages during the selection processes. To efficiently reduce insert-free phages when panning on intact cells, we increased the washing stringency by lowering the pH of the buffer with citric acid. Under standard washing procedures (pH 7.4), only approximately 73% of recovered phages were insert-free after three rounds of selection. Using stringent washing procedures (pH 5.0), approximately 12% of recovered phages contained no scFv. Using this protocol, we have cloned an antibody fragment from a mouse/human hybridoma cell line directed against the disialoganglioside GD2. This study confirms that selection of phage antibodies on cells is efficiently enhanced by assays augmenting the stringency to remove nonspecific binding phages.

Construction and in vitro evaluation of RFT5(scFv)-ETA', a new recombinant single-chain immunotoxin with specific cytotoxicity toward CD25+ Hodgkin-derived cell lines.

The data of a closed phase I/II trial in patients with resistant Hodgkin's lymphoma indicate promising results using a chemically linked anti-CD25 ricin-A immunotoxin (IT) (RFT5-SMPT-dgA). This IT is based on the high-affinity moab RFT5. Since recombinant DNA technology permits the readier production of large amounts of ITs, we constructed a new RFT5-based fusion toxin [RFT5(scFv)-ETA']. We isolated mRNA from the hybridoma cell line RFT5, synthesized first strand cDNA and performed RT-PCR. Amplified coding regions of the light and heavy chain variable domains were joined together with a synthetic (Gly4-Ser)3 linker. The resulting single chain variable fragment (scFv) was fused to a modified Pseudomonas aeruginosa exotoxin A (ETA') lacking its cell-binding domain I. After IPTG-induced expression in Escherichia coli, the 70 kDa His-tagged fusion protein [RFT5(scFv)-ETA'] was isolated by osmotic shock and sonication under denaturing conditions. The recombinant toxin was purified on a Ni2+-NTA chelating sepharose and eluted with 250 mM imidazole. Pooled protein was renatured, dialyzed and concentrated by precipitation. Binding properties of RFT5(scFv)-ETA' were assessed on the CD25-expressing cell line L540cy by ELISA, immunohistochemistry and FACS analysis. CD25-specific binding was confirmed by immunoprecipitation experiments with recombinant human IL-2 receptor alpha. The in vitro toxicity of the chimeric protein was tested on the Hodgkin-derived cell lines L540cy, L428, L1236, a monocyte cell line U937 and a Burkitt lymphoma cell line BL38. RFT5(scFv)-ETA' inhibited protein biosynthesis of L540cy and L428 cells by 50% at concentrations (IC50) of 18 and 12 ng/ml, respectively. CD25-specific toxicity was confirmed by competitive toxicity assays. These data confirm for the first time binding specificity and toxicity of a recombinant anti-CD25 immunotoxin, against Hodgkin-derived cell lines; its applicability on Hodgkin's lymphoma needs yet to be evaluated in vivo.

Construction of phage display libraries from reactive lymph nodes of breast carcinoma patients and selection for specifically binding human single chain Fv on cell lines.

The display of recombinant antibody fragments on the surface of filamentous phage mimicks B cells and is therefore a technology ideal to generate antibodies against any potential target antigen in vitro. In order to obtain tumor specific, high-affinity single chain antibody fragments (scFv), it has been speculated that lymph node tissue from cancer patients infiltrated with activated B cells must be a valuable source of antibody V-genes. The aim of this study was to generate a human scFv-phage library from lymph nodes of patients with breast cancer and to develop a stringent depletion and selection protocol in order to isolate specific single chain antibodies recognizing potentially new antigens in breast cancer. The amplification of the V-genes cloned from regional lymph node tissue and their assembly to single chain variable fragments was optimized in terms of library size and diversity. A large set of degenerated primers, annealing to all known V-gene families, was designed and used under optimized PCR conditions. The amplified V-genes were genetically fused in all possible combinations and cloned into a phagemid vector. Depletion and selection on mammary epithelial and primary breast carcinoma cell lines, respectively led to the isolation of a breast cancer cell line specific scFv (BCK-1 scFv) from this patient-derived scFv-phage display library as demonstrated in polyclonal and monoclonal ELISA, using immobilized cell membrane fractions of the indicated cell lines. A new recombinant breast cancer cell line specific antibody based on V-genes derived from reactive B-lymphocyte-infiltrated lymph nodes of patients with breast cancer was isolated via phage display, performing stringent depletion and selection protocols. We believe that this combination of antibody V-gene source and elaborated phage display depletion and selection strategy will be successful for the retrieval of numerous other recombinant, tumor specific antibody fragments.

An anti-GD2 single chain Fv selected by phage display and fused to Pseudomonas exotoxin A develops specific cytotoxic activity against neuroblastoma derived cell lines.

Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.

Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model.

Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.

Fcgamma receptor 1 (CD64), a target beyond cancer.

Immunotoxins are powerful tools to specifically eliminate deviated cells. Due to the side effects of the original immunotoxins, they were only considered for the treatment of cancer as in these cases, the potential favourable effect outweighed the unwanted toxic side effects. Over time, many improvements in the construction of immunotoxins have been implemented that circumvent, or at least strongly diminish, the side effects. In consequence this opens the way to employ these immunotoxins for the treatment of non-life threatening diseases. One such category of disease could be the many chronic inflammatory disorders in which an uncontrolled interaction between inflammatory cells leads to chronicity. In several of these chronic conditions, activated macrophages, which are characterised by an increased expression of CD64, are known to play a key role. In this review we discuss the data presently available on elimination of activated macrophages through CD64 immunotoxins in several animal models for chronic disease. A chemically linked complete antibody with the plant toxin Ricin-A, proved very effective and provided proof of concept. Subsequently, the development towards genetically engineered, fully human, multivalent single chain based immunotoxins that have diminished immunogenicity, is discussed. The data show that the specific elimination of activated macrophages through CD64 is indeed beneficial for the course of disease. As opposed to other methods used to inactivate or eliminate macrophages, with the CD64 based immunotoxins only the activated population is killed. This may open the way to apply these immunotoxins as therapeutics in chronic inflammatory disease.

In contrast to specific B cells, human basophils are unaffected by the toxic activity of an allergen toxin due to lack of internalization of immunoglobulin E-bound allergen.

BACKGROUND: Specific immunotherapy is the only curative therapy for type I allergies and the alarming increase in allergy prevalence emphasizes the need for additional/alternative strategies for curative treatment. Allergen toxins (AT), fusion products of an allergen with an apoptosis inducing cytotoxin, are a new kind of immunotoxin. OBJECTIVE: AT should allow allergen-specific targeting and elimination of allergy-relevant cells, with B cells being the primary target. An important question is the fate of the effector cells, e.g. mast cells and basophils, which carry allergen-specific IgE: the immunotoxin might even prove to be harmful. METHODS: We established a reliable in vitro B cell model (using two mouse hybridoma cell lines) for testing specificity and toxicity of P5-ETA', a fusion protein of the major timothy grass pollen allergen Phl p 5b and truncated Pseudomonas Exotoxin A. In a second step, we investigated the impact of the AT on human basophils. RESULTS: P5-ETA' reliably eliminated Phl p 5-specific cells in the in vitro B cell model, leaving unspecific B cells unharmed. Human basophils of grass pollen allergic donors specifically bound P5-ETA', released IL-4 and up-regulated the activation marker CD203c, but were not subject to the toxic effect because of lack of internalization of IgE-bound allergen. CONCLUSION: According to our data, basophils are pure effector cells in the context of IgE-bound allergen and not involved in classical antigen presentation.

Determining the linkage of disease-resistance genes to molecular markers: the LOD-SCORE method revisited with regard to necessary sample sizes.

Some approaches to molecular marker-assisted linkage detection for a dominant disease-resistance trait based on a segregating F2 population are discussed. Analysis of two-point linkage is carried out by the traditional measure of maximum lod score. It depends on (1) the maximum-likelihood estimate of the recombination fraction between the marker and the disease-resistance gene locus, (2) the observed absolute frequencies, and (3) the unknown number of tested individuals. If one replaces the absolute frequencies by expressions depending on the unknown sample size and the maximum-likelihood estimate of recombination value, the conventional rule for significant linkage (maximum lod score exceeds a given linkage threshold) can be resolved for the sample size. For each sub-population used for linkage analysis [susceptible (= recessive) individuals, resistant (= dominant) individuals, complete F2] this approach gives a lower bound for the necessary number of individuals required for the detection of significant two-point linkage by the lod-score method.

Theoretical studies on the necessary number of components in mixtures : 4. Number of components and juvenile-mature correlations.

Theoretical studies on the necessary numbers of components in mixtures (for example multiclonal varieties or mixtures of lines) have been performed according to the relations between the juvenile-mature correlations of mixtures and their number of components. For the juvenile-mature correlation rE based upon the values of the single components (= component means at juvenile and mature ages) and the juvenile-mature correlation rM based upon the means of mixtures of different components we usually will have rM>rE. Furthermore, rM will increase with an increasing number of components in the mixtures. The effectiveness of an early selection will be mainly determined by the magnitude of the juvenile-mature correlation. If we have rM>rE an improvement of early testing can be realized by using mixtures instead of single components. But, what are the necessary numbers of components so that rM will be sufficiently high to enable an effective early selection of mixtures? Some relations between rE and rM can be obtained and conclusions have been derived.The statistical approach 'significant difference between rE and rM for a given numerical value of rM' leads to estimates for the necessary number n of components dependent on rM, α, rE and N where: N = total number of components, which are available for the composition of mixtures and α = error probability. For different tree species rE can be estimated by an appropriate formula which depends on T with T = time (in years) from planting date until the mature age.Lambeth's formula, for example, has been developed for height growth in pines. For this situation numerical calculations are performed using rM=0.90 and α=0.05. The necessary numbers n for T=5, T=10, T=20 and T=50 are: 6, 9, 10 and 12 (for N=50); 13, 17, 20 and 23 (for N=100); 26, 34, 40 and 46 (for N=200); 38, 51, 60 and 69 (for N=300); 64, 85, 100 and 114 (for N=500) and 128, 171, 199 and 228 (for N=1,000). The dependence of these necessary numbers n of components on different type I errors α and different levels of rM have been investigated numerically.

Theoretical studies on the necessary number of components in mixtures : 2. Number of components and yielding-ability.

Theoretical studies on the necessary number of components in mixtures (for example multiclonal varieties or mixtures of lines) have been performed according to yielding ability. All theoretical investigations are based upon a Gram-Charlier frequency distribution of the component means with skewness γ1 and kurtosis γ2. The selected fraction p of the best components constitutes the mixture under consideration. The same selection differential S = S (p, γ1, γ2) can be realized by different parameter values of p, γ1 and γ2. Therefore, equal yield levels of the mixture can be achieved by different selected fractions p which implies different numbers of components in the mixture. Numerical results of S = S(p) for different values of γ1 and γ2 are presented and discussed. Of particular interest are the selected fractions p which lead to a maximal selection differential S. These results on S for 'large populations' must be reduced in the case of finite population size. For this correction term we used an approximation B = B (p, n, γ1, γ2) given by Burrows (1972) where n = number of selected components. For given parameter values of γ1, γ2 and p, the necessary number n of components can be calculated by using the condition: Burrows-correction less than a certain percentage g of S - for example with g = 0.05 or g = 0.01. For given γ1 and γ2, the number n leading to a maximal selection differential S can be regarded as necessary number of components (necessary = maximum gain of selection under the given conditions). Numerical results are given for γ2 = 0 and for eight situations which are defined by linear relations γ2 = c γ1 between skewness and kurtosis. These cases will contain all possible numerical situations for γ1 and γ2, which may be relevant for practical applications. The necessary number of components turns out to be nearly independent of the numerical value of the kurtosis γ2. The n-intervals leading to selected fractions p from 0.01 to 0.20 approximately are: 2 ≤ n ≤ 4 for g = 0.05, 6 ≤ n ≤ 20 for g = 0.01 and 11 ≤ n ≤ 40 for g = 0.005, respectively. However, percentages g less than 0.01 would be unrealistically excessive. Therefore, following the assumptions and restrictions given in this paper one may conclude that n = 20 seems to be an appropriate upper bound for the necessary number of components in mixtures.

Theoretical studies on the necessary number of components in mixtures : 3. Number of components and risk considerations.

Theoretical studies on the necessary numbers of components in mixtures (for example multiclonal varieties or mixtures of lines) have been performed according to risk considerations - using the binomial distribution and the Polya-distribution. The 'risk' r of a mixture has been defined as the probability of 'catastrophic' losses (catastrophe = decrease of productivity of q% or more by 'susceptibilities' of the components). Using 1) the binomial distribution and 2) its generalization, the Polya-distribution, and several simplifying assumptions, the risks r = r (x, a, q, n) have been calculated numerically (n = number of components in the mixture, a = parameter for the intensity of contagion and dispersion of 'susceptibilities' (for example: diseases and epidemics), x = probability of 'susceptibility'). The Polya-model reduces to the binomial case if a = 0. The main results are: 1. For each number n of components the risk r decreases markedly with decreasing x (for each q and for each a). 2. For x < = q the risk r decreases with an increasing number n of components (for each a). 3. For each number n of components and x and q with x < q the risk r increases with increasing a. 4. For given q, x and a the functions r = r(n) are asymptotic for larger numbers n of components with n > n(*). In spite of further increasing numbers of components in the mixture the risk remains almost constant. For all situations, where the risk decreases with increasing n these numbers n(*), therefore, can be considered as necessary numbers of components in mixtures, n(*) depends on q, x and a. Nevertheless, a global and rough conclusion can be formulated: In many situations one obtains necessary numbers of 30-40 components for a≠0 and 20-30 components for a = 0.

Estimation of broad sense heritability in plant populations: an improved method.

In the present paper an improved method for estimating broad sense heritability is developed by generalization and improvement of the method of Sakai and Mukaide (1967); this itself is a generalization and improvement of Sakai and Hatakeyama's (1963) modification of Shrikhande's (1957) method to separately estimate the genetic variance, competitional variance and environmental variance of a plant population. Some of the assumptions postulated by these former authors - especially the assumption that the covariance between the genetic and competitional effects equals zero - were omitted. If competitive effects are genetically caused and controlled - and numerous experimental results leave no doubt about this - then this assumption can not be right. Our proposed improvement and generalization avoids this difficulty by considering competition as a usual quantitative character (for each genotype we introduce two quantitative competition-characters: competitive ability and competitive influence). The expected values of the various terms in the variance of plot means (for plots of different sizes) were derived to give a system of simultaneous non-linear equations from which the unknown parameters, genetic variance resp. broad sense heritability, competitional variance, environmental variance etc., can be estimated using least squares methods or direct search methods. The estimates for broad sense heritability are probably more correct and realistic (because of the altered assumptions proposed in this method) than the results of the former authors. The application of the proposed estimation-procedure is demonstrated using Norway-spruce data from Slovakia: Norway-spruce stands (80-90 years old) were investigated for four characters: height, diameter, crown percentage and taper, measured for each single tree.

Maximum likelihood vs. minimum chi-square--a general comparison with applications to the estimation of recombination fractions in two-point linkage analysis.

Some relationships between the estimates of recombination fraction in two-point linkage analysis obtained by maximum likelihood, minimum chi-square, and general least squares are derived. These theoretical results are based on an approximation for the multinomial distribution. Applications (theoretical and experimental) with RFLP (restriction fragment length polymorphism) markers for a segregating F2 population are given. The minimum chi-square estimate is slightly larger than the maximum likelihood estimate. For applications, however, both estimates must be considered to be approximately equal. The least squares estimates are slightly different (larger or smaller) from these estimates.