RT-PCR amplicons in the plasma of multiple myeloma patients--clinical relevance and molecular pathology.

The occurrence of RNA (RT-PCR amplicons) in the plasma of 70 patients was quantified: 65 patients with multiple myeloma (MM), 3 with Waldenstrom's macroglobulinemia (WM), 2 with monoclonal gammopathy of undetermined significance (MGUS), and 50 from healthy controls. A 713nt amplicon occurred in 16/18 MM patients in relapse, 5/8 untreated patients, 2,3 WM patients and 1,2 MGUS plasmas. None of the initial specimens from 37 patients in remission nor the 50 healthy controls was positive. Homology between 4 sequenced 713nt amplicons was > 99.7% and matched (99.6%) a 704nt sequence of the flanking region of the peroxisome proliferator activator receptor gene, located on chromosome 22q11.2. A 255mer within the 713nt amplicon had a 90.2% homology with an AluSc consensus sequence. The occurrence of RNA amplicons seemed to serve as accurate surrogate markers for active disease in MM that provide new insights to the molecular pathogenesis of the disease.

Urine antibody tests: new insights into the dynamics of HIV-1 infection.

BACKGROUND: Noninvasive methodologies provide alternatives to diagnostic blood tests and have high patient acceptance, increased safety, and reduced costs. Such tests may supplement or replace blood diagnostic assays currently in use. METHODS: Using a licensed urine-based test for antibody to HIV-1, we performed 25 991 HIV-1 urine antibody enzyme immunoassay (EIA) screening tests [confirmable by HIV-1 Western blot (WB)] on paired urine and blood specimens obtained from high- and low-risk HIV-1 subjects collected at six sites representative of the US population. RESULTS: Using HIV-1 urine EIA tests confirmed by urine Western blot, a compartmentalized immune response (urine positive/serum negative) occurred in 0.24% of a cohort of 11 896 subjects. In the same cohort, specimens that were urine negative/serum positive occurred in 0.17% of subjects. In a second study of 25 991 subjects that included 859 high-risk individuals, the false-positive urine EIA frequency (urine WB negative or indeterminate) was 1.3%. This false-positive frequency in the high-risk cohort was attributed, in part, to an IgA antibody response. We tabulated urine and serum indeterminate reactivities and examined their possible causes. Data are presented showing that antibodies from a seroindeterminate HIV-1vau group O subject were reactive in urine EIA and urine WB tests. An analysis of the HIV-1vau strain group O env nucleotide sequence disclosed a high frequency of homology with human chromosome 7q31, a fragile site implicated in many human malignancies. CONCLUSIONS: These results demonstrate the utility of urine for alternative HIV-1 antibody testing and provide new insights into the pathogenesis of HIV-1 infection and into potential application of this approach in investigation of other microbial pathogens and toxic compounds.

RNAs in the sera of Persian Gulf War veterans have segments homologous to chromosome 22q11.2.

Reverse transcriptase PCR (RT-PCR) was used for polyribonucleotide assays with sera from deployed Persian Gulf War veterans with the Gulf War Syndrome and a cohort of nonmilitary controls. Sera from veterans contained polyribonucleotides (amplicons) that were obtained by RT-PCR and that ranged in size from 200 to ca. 2,000 bp. Sera from controls did not contain amplicons larger than 450 bp. DNA sequences were derived from two amplicons unique to veterans. These amplicons, which were 414 and 759 nucleotides, were unrelated to each other or to any sequence in gene bank databases. The amplicons contained short segments that were homologous to regions of chromosome 22q11.2, an antigen-responsive hot spot for genetic rearrangements. Many of these short amplicon segments occurred near, between, or in chromosome 22q11.2 Alu sequences. These results suggest that genetic alterations in the 22q11.2 region, possibly induced by exposures to environmental genotoxins during the Persian Gulf War, may have played a role in the pathogenesis of the Gulf War Syndrome. However, the data did not exclude the possibility that other chromosomes also may have been involved. Nonetheless, the detection of polyribonucleotides such as those reported here may have application to the laboratory diagnosis of chronic diseases that have a multifactorial etiology.

Dependence of salt concentration on glycosaminoglycan-lysozyme interactions in cartilage.

The cationic protein, lysozyme, has an extracellular distribution in cartilage but its precise role in this tissue has not yet been established. This study describes the dependence of salt concentration on the binding properties of lysozyme isoforms of different cationic charges, isolated from bovine cartilage, to the two major and structurally similar glycosaminoglycans of cartilage, i.e., chondroitin sulfate and hyaluronan. The binding of most cartilage lysozyme isoforms and hen egg-white lysozyme (control) to chondroitin sulfate and hyaluronan linked to agarose supports displayed optimal levels at approximately 20 and 5-10 mM salt, respectively, but decreased at both lower and higher salt concentrations indicating the electrostatic nature of the interactions. However, optimal binding of the most cationic lysozyme isoform to chondroitin sulfate occurred at 60 mM salt, with significant binding remaining at 150 mM. This isoform also showed binding to hyaluronan up to 60 mM salt, while for the other isoforms binding was observed only up to 150 and 40 mM salt for chondroitin sulfate and hyaluronan, respectively. The low salt concentrations at which these interactions occur are likely to exist in cartilage as shown from equilibrium dialysis studies performed using solutions of chondroitin sulfate (up to 10%, a concentration likely to occur in cartilage). From Scatchard analysis, the affinity of binding of all lysozymes to chondroitin sulfate was similar (Kd = 10(-6) M) and slightly lower than their binding to hyaluronan (Kd = 10(-7) M) of similar molecular mass.

Temporal changes in charge content of cultured chondrocytes from bovine cartilaginous tissues.

The effective charge content of the pericellular matrix of chondrocytes has been determined while the matrix is being synthesized by cells grown in culture for several weeks. The data were compared with estimates determined by chemical analysis. When measurements were performed after digestion of the matrix with papain, there was close agreement between results obtained from both techniques for proteoglycans synthesized by chondrocytes from nasal septum (a non-articular cartilage). By contrast, no such agreement was observed for proteoglycans synthesized by chondrocytes from articular cartilage, even after solubilization of the matrix with papain. While the charge calculated from chemical analysis showed a constant increase with time in culture, that measured by colloid titration showed a cyclical pattern, with maximal values occurring on days 7 and 24 of culture and a minimal value on day 14. This inability to detect all negative groups present in the matrix synthesized by articular chondrocytes would suggest the involvement of these groups in electrostatic interactions. Partial characterization of proteins synthesized by the pericellular matrix indicates that the decrease in charge content observed on day 14 could not be attributed to proteins of a particular molecular mass but possibly to an increase in the total amount of protein present. It is concluded that the marked difference in the availability of negative groups between chondrocytes cultured from articular and non-articular cartilages may reflect differences in the interaction of these negative groups with matrix components; these differences would lead to the distinct structural organization of these two cartilaginous tissues which possess different mechanical functions.

Purification, characterization, and biosynthesis of bovine cartilage lysozyme isoforms.

The cationic protein, lysozyme, has an extracellular distribution in cartilage; however, its biological role in this tissue still remains unclear. This study describes a simple and high yielding procedure for the purification of four novel isoforms of lysozyme from the functionally different articular (metacarpalphalangeal joint) and nonarticular (nasal septum) bovine cartilages. Chromatography of the cartilage extracts on S-Sepharose revealed the presence of four major lysozyme active peaks each of which was further purified to homogeneity by gel filtration and reversed-phase chromatography. Each peak yielded a different molecular mass when analyzed by ion spray mass spectrometry, and material isolated from either cartilage source displayed an identical molecular mass for each lysozyme preparation. N-terminal amino acid sequence and amino acid composition analyses confirmed the presence of four novel lysozyme isoforms in both bovine articular and nonarticular cartilages. The lytic activity of each lysozyme isoform toward Micrococcus lysodeikticus was dependent on both the ionic strength and pH of the buffer, where an increase in activity accompanied an increase in ionic strength. The lysozymes were shown to be synthesized by chondrocytes in vitro, which in addition to the relatively high chemical amounts of lysozyme present in cartilage, would suggest that this small cationic protein has some as yet undetermined biological role within the cartilage extracellular matrix.

Urine-based diagnostic technologies.

The worldwide dissemination of infectious agents has created a demand for simple diagnostic tests. Urine-based testing makes use of non-invasive collection of specimens, and there is no need for expensive facilities and equipment, or for highly trained personnel. As urine antibodies retain activity under normal conditions of transport and storage, such tests appear to have widespread application. Urine-based antibody tests have also indicated a compartmentalized antibody response to HIV-1 infection. Urine studies suggest that antibodies to the products of endogenous viral genes may be involved in the pathogenesis of chronic diseases of suspected viral etiology.

Human endogenous retroviruses: nature, occurrence, and clinical implications in human disease.

Retroviral diagnostics have become standard in human laboratory medicine. While current emphasis is placed on the human exogenous viruses (human immunodeficiency virus and human T-cell leukemia virus), evidence implicating human endogenous retroviruses (HERVs) in various human disease entities continues to mount. Literature on the occurrence of HERVs in human tissues and cells was analyzed. Substantial evidence documents that retrovirus particles were clearly demonstrable in various tissues and cells in both health and disease and were abundant in the placenta and that their occurrence could be implicated in some of the reproductive diseases. The characteristics of HERVs are summarized, mechanisms of replication and regulation are outlined, and the consistent hormonal responsiveness of HERVs is noted. Clear evidence implicating HERV gene products as participants in glomerulonephritis in some cases of systemic lupus erythematosus is adduced. Data implicating HERVs as etiologic factors in reproductive diseases, in some of the autoimmune diseases, in some forms of rheumatoid arthritis and connective tissue disease, in psoriasis, and in some of the inflammatory neurologic diseases are reviewed. The current major needs are to improve methods for HERV detection, to identify the most appropriate HERV prototypes, and to develop diagnostic reagents so that the putative biologic and pathologic roles of HERVs can be better evaluated.

Binding properties of glycosaminoglycans to lysozyme--effect of salt and molecular weight.

The cationic protein, lysozyme, is present in cartilage but its precise role in this tissue has not yet been established. This study shows that two major and structurally similar glycosaminoglycans (GAGs) of cartilage, i.e., chondroitin sulfate (CS) and hyaluronan (HA) interact with lysozyme at salt concentrations up to 40 mM. Such a low salt concentration is likely to occur in cartilage due to exclusion of microions by the high charge density of the proteoglycans (PGs). The affinity of binding to lysozyme increases with increasing molecular weight of HA and is higher for HA (Kd = 1-2 x 10(-8) M and 0.5-1 x 10(-7) M for HA of relative molecular mass of 4 x 10(5) and 5 x 10(4), respectively) than for CS (Kd = 1 x 10(-6) M). The binding displays optimal levels at around 20 mM but decreases at both lower and higher salt concentrations. This dependence of binding on salt concentration resembles that of the enzymic activity of lysozyme for its natural substrate, murein, which is structurally similar to HA/CS. The increase in binding up to 20 mM salt is characteristic for HA/CS-lysozyme interaction as such an effect was not observed in the interaction of heparin with lysozyme or of GAGs with serum albumin. Binding of HA to lysozyme was inhibited by various polyanions but not by uncharged macromolecules, indicating the electrostatic nature of the interaction. The dependence of binding on salt concentration obtained in systems where lysozyme is linked to an agarose support and the GAG is free in solution is similar to that determined when both macromolecules are free in solution; however, the number of GAG disaccharides bound per mole lysozyme increases significantly in the latter system, indicating a marked artifactual steric hindrance effect in the former.

The isolation and properties of whole casein: some implications for comparative studies.

1. Problems in the isolation of whole casein from bovine milk are considered. A summary is given of our experiences in its isolation. 2. The physical characteristics, sedimentation velocity, heterogeneity, absorptivity, nitrogen, phosphorus and carbohydrate contents of whole casein prepared from normal and sub-clinical mastitic milk samples by a variety of methods are compared. The methods are acid precipitation, high-speed centrifugation, with and without added calcium (II), and ammonium sulphate precipitation. 3. A description is given of the low temperature ammonium sulphate procedure preferred for the isolation of whole casein, especially when it is to be used for subsequent fractionation for conformation and micelle studies. 4. The question of the use of bovine casein as a paradigm for the caseins of other mammalian species and the need for further studies of the physical, chemical and biological properties of the caseins are discussed.

The measurement of negative charge content in cartilage using a colloid titration technique.

A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 mu eq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.

Binding of hyaluronan to lysozyme at various pHs and salt concentrations.

Binding of hyaluronan (HA) to lysozyme immobilized on Sepharose-6B was investigated as a function of pH and NaCl concentration. High affinity binding (Kd = 1.0-2.0 x 10(-8) M) was observed at pH 7.5 and at 10-50 mM NaCl; the number of moles of HA bound to lysozyme was twice as high at 30 mM NaCl as at 10 mM. No specific binding was observed at and above 100 mM NaCl. Binding was suppressed in the presence of chaotropic agents such as guanidinium chloride and urea. These results suggest that binding between HA and lysozyme can occur in the extracellular matrix where an electrolyte concentration as low as 50 mM could be expected due to ionic exclusion by the highly negative charge concentration arising from the polyanions present.

Evaluation of nonideality from gel chromatographic partition coefficients. A technique with greater versatility than equilibrium dialysis.

Frontal gel chromatography has been used to measure partition coefficients which enable a quantitative evaluation of the thermodynamic nonideality of small solutes generated by the presence of high concentrations of macromolecular solutes. Equivalence of results obtained by the present method and by equilibrium dialysis is demonstrated in a comparison of results for dextran sulfate-NaCl and dextran-sorbitol systems. Interaction coefficients obtained for dextran-sorbitol and protein-polyethylene glycol 4000 systems yields results which are in reasonable agreement with those predicted on the statistical-mechanical basis of excluded volume. Because of its greater versatility in regard to the range of systems that may be studied, the frontal gel chromatographic procedure is likely to be of particular value for the quantitative characterization of thermodynamic nonideality arising from excluded volume effects in concentrated mixtures of macromolecular solutes.

Basal lamina redevelopment in tumours metastatic to brain:an immunoperoxidase study using an antibody to type-IV collagen.

The basal lamina in a variety of metastatic tumours in brain was assessed with an antibody to type-IV collagen and the indirect immunoperoxidase technique. The antibody was raised in rabbits against type-IV collagen isolated from human placental tissue. Basal lamina redevelopment was demonstrated around individual melanoma cells, between melanoma cells and cerebral parenchyma, and at the tumour-stroma interface in both metastatic melanoma and metastatic carcinoma. At the periphery of metastatic carcinomatous deposits, no basal lamina was observed between tumour cells and the adjacent cerebral parenchyma. Basal lamina staining other than that of cerebral vessels was absent in deposits of poorly differentiated tumours which were unaccompanied by the development of a tumour stroma. It is concluded that metastatic tumours retain the ability to produce basal lamina and, in the case of metastatic epithelial tumours, the redevelopment of basal lamina is dependent on interaction with mesenchymal tissue. The stromal dependency of basal lamina formation by metastatic epithelial tumours suggests the reactivation of a control mechanism acting in normal tissue. Although basal lamina formation in metastatic melanoma can occur in the absence of mesenchymal tissue, there may be some interaction between tumour cells and stroma in the redevelopment of basal lamina at the tumour-stroma interface.

Analysis of collagen from normal and abnormal human bone.

Using cyanogen bromide digestion of bone collagen, we have studied the type of collagen in normal bone, and in samples from patients with Paget's disease of bone, osteomalacia, osteoporosis and renal osteodystrophy. Despite extensive extraction with ethylene diamine tetraacetic acid and guanidine hydrochloride, not all of the glycoproteins could be removed from bone collagen. Upon electrophoresis on sodium dodecyl sulphate polyacrylamide gels, the cyanogen bromide peptides of insoluble bone collagen showed the pattern of type I collagen only in all samples, both normal and abnormal. Amino acid composition of the insoluble collagens and the ratio of hydroxylated and non-hydroxylated amino acids were similar for all adult bone samples. These results suggest that the type of collagen in bone is not altered in the bone diseases examined.

Distribution and lysis of weakly and strongly immunogenic malignant lymphoid cells in normal and immune mice: effects of prior exposure to heat or X-irradiation.

With the use of cells doubly labeled with 51Cr and 125I, an analysis was made of the distribution and lysis in normal and immune syngeneic C58 mice of weakly immunogenic malignant lymphocytes (Ib) and a highly immunogenic variant (IbN) derived from them. The results showed that 51Cr release was not a valid measure of tumor cell lysis in vivo. Prior exposure to 55 degrees C for 30 minutes caused Ib to lyse immediately after iv injection. Prior X-irradiation (10,000 R) enhanced the lysis of Ib in vivo but had only minor effects on IbN. In normal mice 125I-labeled Ib were entrapped in the lungs (approximately to 95%) immediately after iv injection, released in a diphasic manner, and then accumulated in the liver, spleen, bone marrow, and lymph nodes. In immune mice initial entrapment of Ib in the lungs was only about 42%, and label did not accumulate in the described organs. Heat-inactivated Ib were not retained to a significant degree in any of the organs of normal or immune mice. IbN were retained initially (2 hr) at values lower than Ib in the whole body, lungs, and livers of normal mice. 125I-labeled Ib were used to analyze how they were distributed and lysed in normal and immune mice during the first 6 hours after iv injection. To account for the loss of 125I from a tissue because of cell lysis (as distinct from cell redistribution), the amount of 125I associated with cells and blood plasma, and lost from the whole body, was quantified. A significant loss of 125I from the whole body began at 2 hours and continued at a linear rate thereafter. Non-cell-associated 125I occurred in the blood plasma at 1-2 hours and increased at a linear rate thereafter. These results made it possible to distinguish between loss of 125I from a tissue because of cell lysis as distinct from the redistribution of intact labeled cells.

The basal lamina in basal cell carcinoma, Bowen's disease, squamous cell carcinoma and keratoacanthoma: an immunoperoxidase study using an antibody to type IV collagen.

The basal lamina in a variety of skin tumours was assessed with an antibody to type IV collagen and the peroxidase-antiperoxidase (PAP) technique. The antibody was raised in rabbits against type IV collagen isolated from human placental tissue. The basal lamina in Bowen's disease was essentially intact while squamous cell and basal cell carcinomas showed focal loss in areas of tumour cell invasion. However, both tumours showed preservation of basal lamina around the majority of projections and nests of tumour within the dermis. Many keratoacanthomas showed extensive loss of the basal lamina. This loss appears associated with advanced keratinization at the base of the lesion and may represent an involutional change possibly secondary to inflammation. It is concluded that epidermal tumour cells following local invasion may cease migration and produce a distinct continuous basal lamina similar to that of the normal dermo-epidermal junction. Loss of basal lamina appears restricted to foci of ongoing tumour invasion.