A reply to "Relevant factors in the eutrophication of the Uruguay River and the Río Negro".

A recent paper by Beretta-Blanco and Carrasco-Letelier (2021) claims that agricultural eutrophication is not one of the main causes for cyanobacterial blooms in rivers and artificial reservoirs. By combining rivers of markedly different hydrological characteristics e.g., presence/absence and number of dams, river discharge and geological setting, the study speculates about the role of nutrients for modulating phytoplankton chlorophyll-a. Here, we identified serious flaws, from erratic and inaccurate data manipulation. The study did not define how erroneous original dataset values were treated, how the variables below the detection/quantification limit were numerically introduced, lack of mandatory variables for river studies such as flow and rainfall, arbitrary removal of pH > 7.5 values (which were not outliers), and finally how extreme values of other environmental variables were included. In addition, we identified conceptual and procedural mistakes such as biased construction/evaluation of model prediction capability. The study trained the model using pooled data from a short restricted lotic section of the (large) Uruguay River and from both lotic and reservoir domains of the Negro River, but then tested predictability within the (small) Cuareim River. Besides these methodological considerations, the article shows misinterpretations of the statistical correlation of cause and effect neglecting basic limnological knowledge of the ecology of harmful algal blooms (HABs) and international research on land use effects on freshwater quality. The argument that pH is a predictor variable for HABs neglects overwhelming basic paradigms of carbon fluxes and change in pH because of primary productivity. As a result, the article introduces the notion that HABs formation are not related to agricultural land use and water residence time and generate a great risk for the management of surface waterbodies. This reply also emphasizes the need for good practices of open data management, especially for public databases in view of external reproducibility.

Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance.

The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health problem. Many of the specific gene mutations that cause drug resistance in M. tuberculosis are point mutations. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic method to recognize point mutations in genes associated with isoniazid and rifampin resistance in M. tuberculosis. Specific point mutation-containing sequences and wild-type sequences of cloned mycobacterial genes were PCR-amplified, denatured, and hybridized with PNA probes bound to microplate wells. Using 15-base PNA probes, we established the hybridization temperatures (50 degrees C-55 degrees C) and other experimental conditions suitable for detecting clinically relevant point mutations in the katG and rpoB genes. Hybridization of PCR-amplified sequences that contained these point mutations with complementary mutation-specific PNAs resulted in significant increases in ELISA response compared with hybridization using wild-type-specific PNAs. Conversely, PCR-amplified wild-type sequences hybridized much more efficiently with wild-type PNAs than with the mutation-specific PNAs. Using the M. tuberculosis cloned genes and PCR-PNA-ELISA format developed here, M. tuberculosis sequences containing point mutations associated with drug resistance can be identified in less than 24 h.

Role of serology in the diagnosis of Lyme disease.

Numerous concerns regarding the potential for misdiagnosis of Lyme disease using commercial assays have been voiced by the US Food and Drug Administration (FDA). We attempted to clarify the clinical value of serologic testing for Lyme disease using the results of commonly marketed assays for detecting antibody to Borrelia burgdorferi, the organism that causes Lyme disease. We reviewed published studies on B burgdorferi test performance published through 1998, package insert labeling from FDA-cleared test kits for B burgdorferi, and Lyme Disease Survey Set LY-A from the College of American Pathologists. We assessed the sensitivity and specificity of commercial serologic tests (enzyme-linked immunosorbent assay [ELISA], immunofluorescence antibody [IFA], and immunodot) for diagnosis of Lyme disease. To reduce this risk of misdiagnosis, it is important that clinicians understand the performance characteristics and limitations of these tests. These tests, in common use in clinical or commercial laboratories, should be used only to support a clinical diagnosis of Lyme disease, not as the primary basis for making diagnostic or treatment decisions. Serologic testing is not useful early in the course of Lyme disease because of the low sensitivity of tests in early disease. Serologic testing may be more useful in later disease, at which time sensitivity and specificity of the test are improved. Positive or equivocal results on an ELISA, IFA, or immunodot assay requires supplemental testing with a Western blot assay. A negative result on the Western blot or ELISA indicates that there is no serologic evidence of infection by B burgdorferi at the time the sample was drawn.

Issues and perspectives on the biocompatibility and immunotoxicity evaluation of implanted controlled release systems.

Over the past decade, significant advances and discoveries in cell and molecular biology and biomaterials have provided a foundation for the research and development of new, complex controlled release systems. Many of these new controlled release systems utilize active biological components, i.e., proteins and cells, to achieve their intended therapeutic goal. Utilization of bioactive biological materials in implantable controlled release systems has prompted a broad as well as an in-depth interest in the safety and efficacy of these systems. This short review is intended to provide individuals with a perspective on standards and guidance documents which specifically address biological and immunotoxicity response evaluation for safety of controlled release systems from a regulatory point of view.

Silicone breast implants and autoimmune disease.

In 1992, the Food and Drug Administration requested a voluntary moratorium on the scale and implantation of silicone-gel-filled breast implants because of growing concern over the lack of scientific and clinical data supporting their safety and effectiveness. Breast implants had been reported to cause serious local complications, and new questions about breast implants and increased risk for autoimmune disease, including the rare but sometimes fatal connective tissue disease scleroderma, were also raised. Since that time, clinical studies have focused on the adjuvant effect of silicone and of potential autoantibody production. Epidemiologic studies have ruled out a large increased risk for connective tissue disease overall in women with breast implants, but samples were too small to rule out an increase in rare connective tissue diseases. Nor were studies properly designed to address whether an atypical syndrome might develop in women with breast implants. Meta-analyses of these studies cannot remedy their underlying methodologic weaknesses. While the question of whether rare connective tissue disease is associated with breast implants may never be answered definitively, recent progress in identifying new syndromes such as fibromyalgia and chronic fatigue syndrome may provide an insight into methodology for evaluating the existence of a silicone-related syndrome in women with breast implants.

Acquisition and generalization of social skills by high school students with mild mental retardation.

Initial acquisition of social skills by high school students with mild mental retardation was measured during the social skills training game Stacking the Deck. Generalization probes were conducted outside of the training setting (immediately preceding and following training, and 6 weeks posttraining). Due to the high degree of dissimilarity between stimuli in and outside of the training setting, we hypothesized that generalization of skills to nontraining environments would be minimal. Two replications of an initial single-case study were conducted. Students demonstrated acquisition of social skills across game conditions. Immediate generalization of trained social skills did not occur. A possible "deferred generalization" effect was evidenced at 6 weeks posttraining. Results were discussed with respect to the "integrity of common stimuli" and opportunities to respond.

Differential permeability of the blood-tumour barrier in intracerebral tumour-bearing rats: antidrug antibody to achieve systemic drug rescue.

The feasibility of utilizing the differential permeability of the blood-tumour barrier to low- vs. high-molecular-weight compounds is demonstrated in a brain tumour model. Nude rats (n = 27) with or without intracerebral tumours received intravenous [3H]methotrexate (M(r) 454), followed 60 min later by antimethotrexate antibody (M(r) 150,000) or nonspecific mouse antibody. Antimethotrexate antibody resulted in 93% binding of serum methotrexate. In contrast, the percentage of antibody-bound methotrexate in brain and intracerebral tumour was only slightly greater than preantibody protein binding. Methotrexate delivery to tumour was significantly greater than to brain adjacent to tumour and normal brain. The percentage delivery of [3H]methotrexate to all areas of brain was similar between animals receiving antimethotrexate antibody and nonspecific antibody. These findings support the theory that a drug rescue method may be developed that may permit the safe administration of increased dosages of chemotherapeutic drugs for the treatment of intracerebral tumours.

Brain nicotinic receptors isolated by a monospecific antibody against a synthetic alpha-3 subunit receptor peptide compared to a monoclonal anti-idiotypic (to nicotine) antibody.

Nicotinic cholinergic receptor proteins purified from rat brain by immunoaffinity chromatography were characterized using the anti-S3 polyclonal antibody vs. the anti-idiotypic monoclonal antibody 422F11 (generated against an antibody to nicotine). Anti-S3 IgG was purified to homogeneity; anti-S3-Sepharose 4B and 422F11-Sepharose 4B each depleted 3H-nicotine binding sites from brain. Nicotinic receptors isolated from both immunoaffinity columns showed major bands (silver-stained) at 55K and 70K. Using anti-S3 serum as probe, Western blots of nicotinic receptors isolated by the two immunoaffinity gels also showed major bands at 55 and 70K. However, Western blots of fresh brain extracts revealed a major band at 80K and minor bands at 55K and 70K. These results show similar nicotinic cholinergic receptor proteins isolated by the anti-S3 and 422F11 anti-idiotypic antibodies; 80K was dominant only when fresh brain extract was subjected to Western blotting without prior immunoaffinity purification.

Tissue localization of methotrexate-monoclonal-IgM immunoconjugates: anti-SSEA-1 and MOPC 104E in mouse teratocarcinomas and normal tissues.

Methotrexate (MTX) was coupled to the tumor-targeting monoclonal IgM, anti-SSEA-1 and the non-targeting myeloma IgM, MOPC 104E. At 24-h intervals following injection, drug deposition in MH-15 teratocarcinomas and in several normal tissues was followed by immunoperoxidase microscopy using the M16 monoclonal antibody to MTX. MTX-anti-SSEA-1 was deposited on the surface and in the interior of living tumor cells 24 h after injection; at 48 h and after, only low-level binding to necrotic tissue was found. There was no significant gradation in staining from the outside to the interior of the tumors. In tumors, the control MOPC 104E immunoconjugate was detectable only in necrotic tissue. Binding to SSEA-1-expressing normal tissues was undetectable, except for pericryptal fibroblasts in the small intestine. No significant pathology was found in normal tissues that are SSEA-1 positive. High levels of the immunoconjugate were detected in the liver, where MTX was found predominantly in Kupffer cells and possibly in hepatocytes; again, no significant morphological changes were associated with this retention. Thus tumor-associated antigens can be suitable targets for antibody-drug conjugates even when present in normal tissues and in large quantities, provided that the antigens in normal tissues are inaccessible. Moreover, deposition in viable tumor tissue can be assessed using monoclonal antibodies to methotrexate.

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.

Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM. A constitutive form of P-450 is also implicated in nicotine metabolism, while purified P-450IA1 and P-450IIC6 show no detectable activity. The lack of P-450IA1 activity substantiates work by others who also failed to observe an increase in the efficiency of nicotine metabolism to cotinine by microsomes from rats that had been pretreated with benzanthracene. This result is in contrast to work with purified rabbit liver enzymes, in which P-450IA1 exhibited low but measurable activity. Our results support the notion that nicotine metabolism to cotinine by P-450 enzymes is highly species dependent. Thus, it is unwise in some cases to extrapolate results obtained by animal model study to the possible role of specific forms of the P-450 enzymes in nicotine metabolism in humans.

Characterization of nicotinic receptors on cultured cortical neurons using anti-idiotypic antibodies and ligand binding.

Monoclonal anti-idiotypic antibodies that represent the internal image of nicotine's natural isomer, L-nicotine, were used in conjunction with L-[3H]nicotine binding to characterize nicotinic receptors on neurons cultured from fetal rat cortex. Of the antibodies tested, two (422F11 and 420G11) were found that recognized a class of high affinity [3H]nicotine binding sites present on neuronal cells, but not on glia. The binding properties and pharmacological specificity of these sites compared well with those determined previously for putative nicotinic cholinergic receptors in adult rat brain. The binding of [3H]nicotine to neuronal receptors was effectively inhibited by both antibodies. Receptor-bearing cells were identified using indirect immunofluorescence. Approximately 20 to 30% of the cells were labeled specifically by the anti-idiotypes. Labeling was blocked by L-nicotine and other nicotinic agonists, but not by antagonists or by alpha and neuronal bungarotoxins. The majority of cells which were labeled had either bipolar or pyramidal morphology. Fluorescent labeling was associated with cell bodies as well as with axonal and dendritic processes, consistent with the proposed roles of neuronal nicotinic receptors in neuromodulation and synaptic transmission. The results suggest that anti-idiotypic antibodies may provide a new tool suitable for studying the locations, structure and functional significance of high affinity neuronal nicotinic receptors at the cellular level.

Interaction of cotinine with rat hepatic microsomal P-450. Comparison with metyrapone and immunomodulation of cotinine and metyrapone binding by monoclonal anti-cotinine antibodies.

The ability of the major nicotine metabolite, cotinine, to interact with rat liver microsomal cytochrome P-450 and the immunomodulatory effects of anti-cotinine antibodies were studied. Cotinine induced type II spectral changes with both microsomes from phenobarbital (PB)-induced rats and purified P-450 with apparent Ks values of 97 and 750 microM, respectively. In contrast, the Ks value was 0.3 microM for metyrapone and 5 microM for nicotine with both the microsomes and purified enzyme. The apparent Ki value for cotinine inhibition of 7-pentoxyresorufin O-dealkylase activity with the microsomes (87 microM) was approximately 87- and 870-fold higher than for nicotine and metyrapone, respectively. Monoclonal antibodies produced against cotinine cross-reacted equally well with metyrapone. They specifically blocked enzyme binding of both drugs based on dose-dependent inhibition of spectral changes, and reversed the metyrapone-induced inhibition of microsomal O-dealkylase activity. In contrast, antibodies to nicotine did not cross-react with cotinine or metyrapone and had no effect on their activity, although they did block the action of nicotine. These results demonstrate that cotinine binding to P-450 from PB-induced rats and inhibition of functional activity in vitro are qualitatively like the effects of metyrapone and nicotine, and that monoclonal anti-cotinine antibodies are useful molecular probes of the interactions between cotinine and metyrapone with the enzyme.

Cotinine analytical workshop report: consideration of analytical methods for determining cotinine in human body fluids as a measure of passive exposure to tobacco smoke.

A two-day technical workshop was convened November 10-11, 1986, to discuss analytical approaches for determining trace amounts of cotinine in human body fluids resulting from passive exposure to environmental tobacco smoke (ETS). The workshop, jointly sponsored by the U.S. Environmental Protection Agency and Centers for Disease Control, was attended by scientists with expertise in cotinine analytical methodology and/or conduct of human monitoring studies related to ETS. The workshop format included technical presentations, separate panel discussions on chromatography and immunoassay analytical approaches, and group discussions related to the quality assurance/quality control aspects of future monitoring programs. This report presents a consensus of opinion on general issues before the workshop panel participants and also a detailed comparison of several analytical approaches being used by the various represented laboratories. The salient features of the chromatography and immunoassay analytical methods are discussed separately.

Idiotype-anti-idiotype hapten immunoassays: assay for cotinine.

Practical application of the idiotype-anti-idiotype reaction to hapten immunoassays has been demonstrated with cotinine as an example. The assay relies on the ability of cotinine, a major nicotine metabolite, to inhibit binding between a monoclonal anti-cotinine antibody (the idiotype) and a second monoclonal antibody (the anti-idiotype) specific for the antigen combining region on the idiotype. A solid phase enzyme-linked immunoadsorbent assay (ELISA) format was adopted in which fluid phase anti-cotinine and cotinine present either as a standard or in a test sample were incubated in microtiter plate wells coated with F(ab')2 fragments of the anti-idiotype. Horseradish peroxidase-labeled protein A and o-phenylenediamine were used to detect idiotype-anti-idiotype binding. Under optimal assay conditions, 0.9 ng cotinine inhibited immune binding by 50% and as little as 0.04 ng could be detected. In contrast, nearly 70 times more trans-3'-hydroxycotinine, a major urinary metabolite, and over 1000-fold more nicotine were required for 50% inhibition. Several other metabolites and structurally related compounds also were poor competitors. Assay reliability was good over a range of cotinine concentrations from 5 to 500 ng/ml saliva with intraassay coefficients of variation between 6 and 10% and interassay values between 6 and 13%. Also, there was a strong correlation (R2 = 0.994) between the cotinine levels found in saliva from 35 cigarette smokers with the idiotype-anti-idiotype assay and a cotinine-anti-cotinine ELISA. Because only monoclonal antibodies and antigen are required, the idiotype-anti-idiotype immunoassay offers a high degree of standardization without the need to prepare labeled hapten derivatives or macromolecular conjugates for solid phase assays.

Anti-idiotypic antibody probes of neuronal nicotinic receptors.

Monoclonal anti-idiotypic antibodies specific for the combining site on a monoclonal antinicotine were used in immunocytochemistry to localize nicotine binding sites on rat brain cortical sections and in immunoaffinity chromatography to isolate receptor from solubilized brain tissue. The receptor, which consists of two subunits with Mr values of 43 and 50 kDa, was eluted from the antiidiotype column with either pH3 citrate buffer or 25 mM (-)-nicotine, but was not present in eluates from immobilized anti-Electrophorus acetylcholine receptor or anti-methotrexate. The anti-idiotypes specifically inhibited [3H]nicotine binding to rat brain homogenate and (-)-nicotine inhibited anti-idiotype binding to brain sections based on abrogation of immunofluorescence staining. These results are consistent with the operational definition of the anti-idiotypes as the internal image of nicotine, and demonstrate their value as immunochemical probes of nicotinic receptors.

Dissociation between murine spleen cell mitogenic activity of enterotoxin contaminants and anti-tumor activity of staphylococcal protein A.

Soluble staphylococcal protein A (SpA) in the form of high m.w. complexes with IgG has been shown to significantly inhibit the growth of Meth A fibrosarcomas in BALB/c mice. Although SpA reportedly is a potent T cell mitogen that can induce immune cell proliferation and production of humoral factors with anti-tumor activity, it has been suggested that mitogenic enterotoxin contaminants might be responsible for these effects. The purpose of the present study was to investigate the nature of SpA-induced cell proliferation and the relationship between mitogenicity and the anti-tumor effect that we observed in our mouse model. SpA stimulated the proliferation of a mixed population of splenic B and T cells from BALB/c mice, but activity did not require the presence of IgG in the culture medium. Furthermore, mitogenic activity could be inhibited completely by anti-SEA plus anti-SEB, but was unaffected by anti-SpA. HPLC-purified SpA was inactive while the mitogenic factor(s) had the same retention time as authentic enterotoxin and its activity was inhibited by anti-SEA and anti-SEB, but not by anti-SpA. Enterotoxin-free rSpA produced in Escherichia coli had the same IgG binding capacity as the staphylococcal product but was not mitogenic. These data indicate that SEA and SEB completely account for mitogenicity in SpA preparations. In contrast, we found that optimal concentrations of rSpA as well as crude and HPLC purified staphylococcal SpA were equally effective in inhibiting the growth of established Meth A fibrosarcomas demonstrating that SpA is responsible for antitumor activity without any apparent role for enterotoxins.