Demonstrating reliability through transparency: A scientific validity framework to assist scientists and lawyers in criminal proceedings.

In recent decades, forensic science evidence has come to play an increasingly significant role in criminal proceedings. However, the ability of non-scientists (lay-persons, including lawyers and judges) within criminal justice systems to recognise and resolve issues of validity and reliability relating to expert opinion evidence has not maintained pace with the need to do so. Despite international scrutiny from scientists, statisticians, governments and those involved in law reform, the parameters of a) different forensic disciplines and b) some case specific interpretations, remain elusive to some legal practitioners and judges. It is therefore essential that within the context of national, and increasingly international and transnational criminal investigations, forensic science experts convey the evidential value of the scientific findings in a manner that is understandable to, and useable by all. To assist, this paper first identifies the organisational structures necessary to scaffold and support the delivery of reliable expert opinion evidence. This is followed by a format for transparently reporting the reasoning and the scientific validity underpinning the expert's evidence within their report: a tripartite Scientific Validity Framework. This framework is comprised of (i) foundational validity, (ii) applied validity and (iii) the new concept of evaluative validity. Such a framework, because of its underlying scientific principles, is applicable to expert reports in any jurisdiction and is complementary to different national approaches. That is because utilising this framework could ensure that experts can, and do, demonstrate that their case-specific opinion is reliable and alert the legal profession to the expert's reasoning process and any limitations in the scientific validity underpinning the opinion.

Interferon responses in schizophrenia and major depressive disorders.

The spontaneous and induced interferon (IFN) production in whole blood cultures was examined in 45 psychiatric inpatients and in 65 normal controls. Among inpatients there were 32 who were chronic schizophrenics (14 women, 18 men) and 13 who were severely depressed (11 women, 2 men). The analysis of the pooled results of assays in the heterogeneous population showed that leukocytes of the psychiatric patients produced significantly lower levels of IFN after stimulation with virus (NDV), lipopolysaccharide (LPS), and IFN spontaneously released without the inducers that control cells. In contrast, there was no difference between the psychiatric patients and controls in IFN response to phytohemagglutinin and phorbol myristate acetate (PHA + PMA). The results apparently confirmed observations made by Moises et al (1985) and Katila et al (1989). We have also tested our hypothesis that the statistics may mask the individual pattern of IFN response related to the specific psychiatric diagnosis, however. In fact, in the group of chronic schizophrenics we have found either high or low responders to all IFN inducers (NDV, PHA + PMA and LPS). Furthermore, the patients with high IFN response had dominant positive symptoms of schizophrenia (delusions, hallucinations, bizarre behavior and thought disorder). Whereas, in the patients with low IFN response the negative symptoms prevailed (asociality or withdrawal, flat affect, attention impairment, abolition or apathy). In plasma samples of schizophrenics, factors were detected that transferred a hypersensitivity to the IFN inducers to normal donor leukocytes. For instance, in leukocytes cultured in the presence of plasma from schizophrenics, there were 71% of high IFN responders after stimulation with NDV, versus 26% of high IFN responders in the presence of plasma from normal controls. We suggest that the factors may belong to the class of opioid peptides, which interact with the production of cytokines including IFNs.

Synergistic action of interferons (IFN-alpha/beta and IFN-gamma) of different species origin.

The effects of the mixtures of human, porcine and murine interferons (IFNs) alpha, beta and gamma and bovine IFN-alpha were studied in human, porcine and murine cells. The synergistic action of the mixtures of IFN-alpha/beta and IFN-gamma occurred not only in the homologous but also in the heterologous systems. The synergistic effect was observed when one of the IFNs was of alpha/beta type and the other one of gamma type and when both of them were active in the target cells, regardless of their species origin. Because PoIFN-alpha is active in the cells of numerous mammalian species, it is a good tool for the studies on the synergistic effects. The synergistic action of the mixture of IFNs was abolished after inactivation of one of the IFNs. Specific inactivation of IFN-alpha/beta in the mixture was accomplished by incubation with anti-IFN sera and in the case of IFN-gamma, by acidifying to pH 2.

Human acid-labile interferon alpha.

In acquired immune deficiency syndrome (AIDS) and autoimmune diseases, like systemic lupus erythematosus (SLE), persisting high levels of interferon (IFN) are detectable in plasma. The IFN has been identified as an unusual human acid-labile IFN-alpha (al-IFN-alpha). Its properties are similar to that of the known alpha interferons except sensitivity to acid treatment (pH 2) which is characteristic for IFN-gamma. The nature of al-IFN-alpha is not known. Four hypotheses have been presented that suggested that: a) al-IFN-alpha may be a product of a distinct IFN gene; b) or a posttranscriptionally modified IFN-alpha molecule; c) the phenomenon of al-IFN-alpha may be an effect of synergistic action of a mixture of acid-stable IFN-alpha and acid-labile IFN-gamma; d) or the effect may be a result of an interaction of acid-stable IFN-alpha with an unknown factor(s) present in plasma and associated with progression of HIV infection or autoimmune diseases. Neither of the hypotheses have been experimentally proven. To verify the most probable hypothesis of the synergistic interactions between the acid stable and labile IFNs, the experiments with the artificial mixtures of nHuIFN-alpha and rHuIFN-gamma were performed. The synergistic effect was abolished by the treatment either with pH 2 or with anti-IFN-gamma antibodies. The residual activity was similar in both cases and corresponded to acid-stable IFN-alpha. However, al-IFN-gamma (AIDS serum with high IFN level) was found to be much more sensitive to acid treatment and only negligible effect of anti-IFN-gamma serum was observed. It suggests that the synergistic effect may be only slightly responsible for the phenomenon of acid lability of IFN-alpha. For explanation of the occurrence of al-IFN-alpha the hypothesis of existence of a factor(s) interacting with IFN-alpha should be taken into consideration.

Effects of interferons, interferon inducers and growth factors on phagocytosis measured by quantitative determination of synthetic compound ingested by mouse bone marrow-derived macrophages.

Sodium salt of 9-oxo-10-acridineacetohydroxamic acid (HCA), a new synthetic compound, forms small crystals in aqueous solution. These crystals were easily phagocytized by the mouse bone marrow-derived macrophages. The ingested HCA crystals were visible under light microscope as dark granules. The degree of phagocytosis was estimated by the spectroscopic measurements of absorption of ingested HCA. About 10 day-old cultures of mouse bone marrow-derived macrophages were found to be suitable for a study on the effect of murine or human interferons. It was observed that murine interferons alpha, beta and gamma at low concentrations (10-200 U/ml) stimulated and higher concentrations (400-1000 U/ml) had no effect on the phagocytosis. Previous treatment of interferons with anti-IFN sera abolished the effect of the interferons. CMA-induced interferon and growth factors were found to modify the phagocytic activity of macrophage cultures.

A simple colorimetric method for the measurement of phagocytosis of crystals of low molecular weight compound by macrophages.

Sodium salt of 9-oxo-10-acridineacetohydroxamic acid (HCA), a new synthetic compound, forms small crystals in aqueous solutions. These crystals were easily phagocytable by the mouse bone marrow-derived macrophages. The ingested HCA crystals were visible under light microscope as dark granules. The counting of cells with the granules allowed the calculation of number of the phagocytic cells in the preparation. It was also possible to measure the amount of ingested HCA by the cells using colorimetric method. The kinetics of phagocytosis of HCA showed that the process was rapid. Quantitative measurements of ingestion of HCA reflected the phagocytic activity of the cultured cells.

Tolpa Torf Preparation (TTP) induces interferon and tumor necrosis factor production in human peripheral blood leukocytes.

Tolpa Torf Preparation (TTP) is a natural immunomodulating drug registered in Poland for use in humans. TTP is a biologically active low molecular weight fraction of an extract from peat containing organic substances, primary bound sugars, amino-acids, uronic and huminic acids and mineral salts. The toxicity of TTP is remarkably low, eg. cytotoxicity (CD50) for human peripheral blood leukocytes (PBL) is 1-9 mg/ml. We have discovered that TTP is an interferon (IFN) and tumor necrosis factor (TNF) inducer in human PBL. The IFN and TNF response of the PBL cultures was dose dependent. The optimal concentration of TTP for IFN or TNF response was 10-100 micrograms/ml. The cytokines stimulated by TTP were IFN-gamma, IFN-alpha and TNF-alpha. Ten commercial batches of TTP have been found to be active as cytokines inducers although variations in their activities were observed. On the other hand, 8 batches of TTP rejected by the producer because of the inadequate immunostimulating activity determined in mice, were found to be significantly less active than the commercial preparations. Over 115 buffy coats from the individual blood donors were used to prepare PBL cultures for this study. Approximately 20% of the PBL cultures were unresponsive to TTP. The IFN and TNF response of PBL to other inducers: phytohemagglutinin (PHA) or lipopolysaccharides (LPS) also varied. Whereas only 7% of PBL could not be stimulated by PHA, as much as 20-50% of PBL failed to produce IFN or TNF when treated with LPS. We suggest that TTP may have clinically useful activities connected with the capacity of stimulation of IFNs and TNF production.

Decrease of enhanced interferon alpha levels in sera of HIV-infected and AIDS patients receiving combined antiretroviral therapy.

In the advanced stages of human immunodeficiency virus (HIV) infection the defective interferon (IFN) responses have been observed. Persisting high lebels of the acid-labile interferons (al-IFNs) have been found in sera of the patients with AIDS. The combined antiretroviral therapy, that included the reverse transcriptase and viral protease inhibitors, resulted in a significant improvement of the clinical state of the majority of HIV-infected patients. In this report we describe the levels of IFNs in 41 HIV patients subjected to the combined treatment. High IFN levels (median 84, up to 576 U/ml) were found in sera of patients classified as the stage C2 or C3 of AIDS before the treatment. The combined therapy resulted in the decrease of IFN levels (median 7.5, up to 24 U/ml) that approached the levels of IFNs detected in the HIV+, A1-A3 stage patients (median 4, up to 36 U/ml). In contrast, the unsuccessful therapy connected with the worsening of the clinical state and the decrease of CD4+ cell count had no effect on the IFNs levels (median 48, up to 96 U/ml). Thus, the measurements of IFN activity in sera may be useful for monitoring the effects of the antiretroviral combined therapy. In sera of the AIDS patients, subjected to the antiviral bioassays, the mixture of the acid-labile and acid-stable form of IFN-alpha, with the prevailing al-IFN-alpha, have been detected.

1,4-naphthoquinone-2-sulfonic acid derivatives coupled to dextran as carriers for interferon.

Three derivatives of 1,4-naphthoquinone-2-sulfonic acid possessing the fragments of Cibacron Blue (CB) were synthesized and bound to Dextran T 2000 by ether binding. Polymers IVD, VD and VID were incubated with interferon (IFN) to obtain complexes: carrier-IFN. It was found that polymer VID has weaker affinity to mouse IFN-beta/alpha and to human IFN-beta than Blue Dextran. The polymers IVD and VD had no affinity to the interferon.

New benzoquinone derivatives of dextran as carriers for interferon.

A series of derivatives of benzoquinone-2-sulfonic acid (I-VI) containing constituents of the structure of Cibacron Blue (CB) was synthesized and bound to dextran T 2000 with ether binding (VII-XII). Two dextran polymers XI and XII showed in biological assays high affinity for interferon (IFN).

Seleno-organic compounds induce interferon and tumor necrosis factor in human but not in rat or mouse lymphoid cells.

The seleno-organic compounds are highly active in several anti-inflammatory assays performed in mice and rats. However, they differ from the classical non-steroidal anti-inflammatory drugs including indomethacin despite the fact that both types of drugs are inhibitors of prostaglandins and leukotrienes. Furthermore, ebselen and analogs are potent anti-oxidants in many animal cell cultures. The toxicity of the drugs is low because selenium in their structure is not bioavailable. We have discovered that the seleno-organic compounds induce interferon gamma (IFN-gamma), IFN-alpha, tumor necrosis factor alpha (TNF-alpha) and other cytokines in human peripheral blood leukocytes (PBL). Furthermore, the action of the drugs and PHA or Con A was synergistic. However, ebselen and analogs were found to be inactive as the cytokine inducers in cultured rat or mouse lymphoid cells. In contrast to their effects in human PBL, the drugs even inhibited the production of IFN-gamma after stimulation with PHA or Con A. The inhibition was dose dependent. We suggest that the induction of IFN by ebselen and analogs is species specific and it may depend on interaction of the drugs with a specific receptor and/or signal-transducing system present in human but not in some animal cells.

New derivatives of blue dextran binding and stabilizing human or mouse interferons.

Mouse or human interferons were found to bind strongly to Blue Dextran 2000 and its eight new derivatives (compound VII to XIV covalently bound to Dextran 2000). The congeners of BD were pale violet or grey. The affinity of Blue Dextran and its congeners to interferons were: IFN-beta greater than IFN-alpha greater than IFN-gamma. The carriers stabilized the antiviral activity of beta-type of interferon during storage at 4 degrees C.

Antigen-specific and nonspecific interferon response of peripheral blood leukocytes from patients with chronic brucellosis.

Thirty seven blood samples from 21 patients with chronic brucellosis were studied for interferon response to Brucella specific antigens and to the classical IFN inducers. Whole blood technique for IFN induction and bioassay with A549 cells challenged with EMC virus for IFN detection were used. Two different antigen preparations (BRU-1 and BRU-2) used for the serologic diagnosis of brucellosis, stimulated significantly (P less than 0.001) the synthesis of IFN-alpha and IFN-gamma in the whole blood cultures from the patients with chronic brucellosis but not from the control subjects. The detoxified antigen (brucellin) was inactive as the IFN inducer. BRU-1 and BRU-2 antigens induced also low levels of IFN-alpha + IFN-gamma in the short term cultures of the separated peripheral blood leukocytes (5-10 X 10(6) cells/ml) from healthy blood donors. This resembled stimulation of the leukocytes with LPS. Brucellin was inactive in the leukocyte culture. Despite the chronic infection lasting many years the brucellosis patients had apparently intact IFN system because the response of their leukocytes to NDV, PHA + PMA or LPS was not significantly different from that of the healthy blood donors. The importance of the relative balance of the IFN system for the pathogenesis of brucellosis is suggested and contrasted with IFN disfunction in the acquired immune deficiency syndrome.

Long-term assessment of interferon responses in the HIV+ and AIDS patients.

Impairment of interferon (IFN) system in human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) became a basis for searching IFN responses to monitor the disease progression. For detailed investigations 16 HIV+/AIDS patients with at least 4 successively taken blood samples available for IFN determinations were selected. IFN responses were tested in two ways. Firstly, IFN level in plasma was measured. Secondly, capacity of IFN production by leukocytes was evaluated. The latter was determined in the whole blood assay, in which Newcastle disease virus (NDV) and phytohemagglutinin (PHA) were used as IFN-alpha and IFN-gamma inducers, respectively. The levels of IFN induced in whole blood leukocytes varied considerably in all individuals that had been tested. Nevertheless, two patterns of IFN responses were observed. In pattern I, patients had low levels of IFN in plasma and high levels of induced IFN-alpha and IFN-gamma. It was characteristic for 8 patients in good clinical condition. On the contrary, severe disease found in 2 patients was correlated with high levels of IFN in plasma and low levels of induced IFNs (pattern II). In 6 patients IFN responses were classified as intermediate pattern I/II suggesting transition from pattern I to pattern II. A variation of pattern I was found in the case of a patient defined as long-term survivor having relatively low levels of all IFN tested. The results suggested that interferon measurements reflected clinical condition of HIV+ patients showing not only past but also current immune changes.

Effects of some lipophilic carboxylic acid on release of interferon gamma from the human peripheral blood leukocytes.

Lipophilic carboxylic acids and their salts were tested for the ability of interferon induction in the cultures of human peripheral blood leukocytes. Of 13 tested organic compounds, two were selected: decyl-malonic acid and sodium salt of octylmethane-tri-(2-oxabutanoic)acid--which stimulated the synthesis of low amounts of gamma interferon.

Simultaneous induction of interferon gamma and tumor necrosis factor alpha by different seleno-organic compounds in human peripheral blood leukocytes.

Ebselen is known as anti-inflammatory and anti-oxidant selenium containing drug. We have synthetized 13 seleno-organic compounds, analogs of ebselen. Seven of them were found to be inducers of interferon gamma (IFN-gamma) and/or tumor necrosis factor alpha (TNF-alpha) in human peripheral blood leukocytes (PBL) cultures. The most active cytokine inducers were: 2-phenyl-1,2-benzisoselenazol-3(2H)-one (1, ebselen), bis [2-(N-phenylcarbamoyl)]phenyl diselenide (7) and bis (2-[N-(2-pyridyl)carbamoyl])phenyl diselenide (8). The amounts of IFN and TNF produced by PBL cultures in response to the seleno-organic compounds were found to be similar to that induced by phytohemagglutinin (PHA). The activities of the seleno-organic compounds were dose-dependent and related to the chemical structure of the drugs suggesting involvement of the specific cytokine-inducer receptor. The simultaneous inductions of IFN-gamma and TNF-alpha were highly correlated, but independent on each other.

Seleno-organic compounds as immunostimulants: an approach to the structure-activity relationship.

Our studies on the seleno-organic compounds were focused at their activities as the modest cytokine inducers in human peripheral blood leukocyte cultures. Our bioassays used in the screening methods were based on the quantitative determinations of mainly two types of cytokines: interferons (IFNs) and tumor necrosis factors (TNFs). More recently we have found that several of the compounds have direct immunotropic actions in vitro and in vivo, in mice and in chickens. The paper summarizes the data related to the cytokine-inducing activity of 65 seleno-organic compounds divided into 4 groups according to their chemical structures. The reference compound was ebselen, the well known experimental drug with various biological activities. Approximately 50% of the compounds were found to be active in our bioassays. The selected compounds induced also IL-6 and GM-CSF. Their activities were clearly correlated with defined chemical structures as well as with the presence of selenium. We suggest that some of the selected by us compounds, other than ebselen, are interesting as immunostimulants and potential antiviral agents and cytokine inducers active in humans.

Interaction of sodium salt of 9-oxo-10-acridineacetic acid (CMA) and its analogs with serum albumin. A model for study on binding of the interferon inducer with receptor.

Equilibrium dialysis, gel filtration and SDS polyacrylamide gel electrophoresis were used to study the interaction of sodium salt of 9-oxo-10-acridineacetic acid (CMA) as well as its analogs 7, 8, 11, 13 - 16 with proteins. The compounds were found to bind mainly to serum albumins. Several other proteins had no affinity to the compounds. The close analogs 7 and 8 (sodium salt of 2,7-dibromo-9-oxo-10-acridineacetic acid and sodium salt of 9-oxo-10-acridinebutyric acid) which were inactive as interferon inducers were found to have greater affinity to bovine, mouse or human albumin than the active IFN inducer--CMA. The mechanism of interaction of CMA as well as its close analogs with albumin resembled the first phase of reaction of pharmacologically active ligands with their specific receptor or acceptor proteins. CMA and some of its close analogs were also shown to stabilize the human erythrocyte membrane against hemolysis in the hypotonic solution. However, the activity of the compounds was much weaker than that of other so called membrane active drugs.

Interferon and tumor necrosis factor responses of HIV+ patients as markers for monitoring of the AIDS progression.

The blood samples taken from 31 HIV+ and AIDS patients were used to study interferon (IFN) and tumor necrosis factor (TNF) responses. The IFN and TNF levels in plasma were determined. In the whole blood assay (whole blood diluted 1:10 with culture medium) Newcastle disease virus (NDV) and phytohemagglutinin (PHA) were used as cytokine inducers. Blood leukocytes of HIV+ patients produced significantly less IFN-alpha after NDV stimulation than the cells of healthy (HIV-) individuals. On the other hand, the production of IFN-gamma in response to PHA was impaired only in AIDS patients with stage CDC IV and CD4+ cell number < 200/microliters. These patients had also increased IFN levels in plasma. Particularly, the high level of IFN in plasma was frequently detected in patients with progressing AIDS with CD4+ cell number < 50/microliters. This type of IFN was identified as a mixture of acid-labile and acid-stable IFN-alpha. The IFN responses of HIV+ patients may be considered as markers for monitoring progression of AIDS and therapy.

Coincidence between spontaneous release of interferon and tumor necrosis factor by colostral leukocytes and the production of a colostrinine by human mammary gland after normal delivery.

We have tentatively identified colostrinines as novel cytokines produced by the mammary gland after delivery and detectable in colostrum. The primary colostrinine, the proline-rich polypeptide, was isolated from ovine colostrum in 1974. It is generally understood that the various factors present in colostrum play a pivotal role in transmitting of passive or active immunity from mother to child. We have found previously that both ovine and human colostrinines are inducers of interferon (IFN) gamma and other cytokines. In this paper, we reported that the leukocytes isolated from human colostrum donated by healthy mothers at 1-9 days after delivery, produced IFNs and tumor necrosis factors (TFNs) spontaneously. The release of IFNs and TNFs coincided with production of a colostrinine that has been isolated from the human colostrum samples and partially characterized. Our results suggest that the maximum production of colostrinine occurs 3 days after delivery. The tolerance (hyporeactivity) of the colostral leukocytes to IFN inducers and the modulation of the TNF response may be the late effects of the colostrinine release.