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Biomedical subjects

M Dinowitz

Publications and source records attributed to M Dinowitz.

At least 19 recordsLinked to original sources
PubMed2002

Viral safety and clearance evaluation--implications for process change and comparability.

A key component in the development of safe cell-based biologicals is the incorporation of procedures to ensure virological safety using a three-pronged approach--raw material testing, including testing of the cell banks; incorporation of virus clearance steps in the production process; and lot-by-lot virus testing of culture harvests. During the development of a biological, changes are made to improve product quality, purity, yield, and consistency and to meet other production requirements. It is important to evaluate process changes in view of virus safety. Changes to the cell culture and purification processes may necessitate the repeat of virus testing and clearance steps to ensure that they have not been adversely affected by the changes. It is also important to evaluate the effect that column re-use has on virus clearance. The impact of process change and column re-use are described and the importance of these at various stages in development and post license is discussed.

Biological Products↗
PubMed1999

Quantitative competitive reverse transcription-PCR as a method to evaluate retrovirus removal during chromatography procedures.

Chinese hamster ovary cells used for pharmaceutical protein production express non-infectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing process to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for validation studies. Some chromatography procedures used for pharmaceutical protein purification utilize low pH (< pH 4.0) elution buffers which readily inactivate X-MuLV. Therefore, cell-based infectivity assays are unable to evaluate the physical removal of X-MuLV by these chromatography procedures. To distinguish viral inactivation by low pH treatment from viral removal by chromatography, a quantitative competitive reverse transcription PCR method capable of quantifying both infectious and non-infectious X-MuLV has been developed. This method quantifies X-MuLV particles in chromatography pools by quantifying the X-MuLV particle RNA (pRNA). The difference between the amount of X-MuLV pRNA in the load pool and the product-containing elution pool represents the extent of X-MuLV removal. This method is an extremely powerful complement to cell based-infectivity assays as it allows physical removal of X-MuLV by chromatography and filtration procedures to be distinguished from X-MuLV inactivation when buffers with the ability to inactivate retrovirus are used.

Animals↗
PubMed1997

Failure of cast immobilization for thumb ulnar collateral ligament avulsion fractures.

To determine if small avulsion fractures of the thumb ulnar collateral ligament (UCL) with minimal (< or = 2.0 mm) displacement can successfully be treated by cast immobilization, the authors reviewed 9 patients with minimally displaced fractures initially treated by casting. Despite immobilization within an average of 2 days of the initial injury (range, 0-6 days), a minimum of 6 weeks of immobilization in a cast, and adequate rehabilitation, all 9 patients had persistent thumb pain, especially with activities requiring strong pinch. After undergoing open reduction and internal fixation, the patients had relief of thumb pain and pinch strength improved from 36% of the contralateral side to 89% (p < .01). Grip strength increased from 77% to 93% (p < .05), but the ranges of motion of the thumb metacarpophalangeal and interphalangeal joints were not significantly altered. Minimally displaced UCL avulsion fractures frequently have significant rotation that prevents successful fracture healing even with prompt cast immobilization.

Adolescent↗
PubMed1993

Genetic stability and recombinant product consistency.

A committee of U.S. industry scientists from the Pharmaceutical Manufacturers Association has reviewed the recent suggestions of Galibert and Center for Biologics Evaluation and Research (CBER) regarding assurances of product consistency by cloning and sequencing efforts. We disagree that their proposals will achieve this goal, and estimate that such efforts will be very costly in terms of regulatory agency time examining artifactual errors as well as industry resources. We feel that current analytical and manufacturing technology is adequate to assure recombinant product consistency without the suggested cloning and sequencing measures.

Animals↗
PubMed1992

Recent studies on retrovirus-like particles in Chinese hamster ovary cells.

Highly concentrated (4000-7000-fold) culture fluids from CHO cells were analysed for the presence of retrovirus-like activity. Concentrates containing reverse transcriptase activity were detected and further purified by sucrose density gradient centrifugation. Particles banding at 1.13-1.16 g/ml were found to contain nucleic acid sequences and structural proteins related to those found in murine and other retroviruses. Analysis of the endonuclease gene has shown no intact open reading frames. Approximately 100-300 copies of the C-type sequences were present in the genome of CHO cell lines as well as in the DNA extracted from Chinese hamster liver, indicating that these sequences are present in the germ line of the species. Concentrates were analysed for infectivity by direct inoculation and co-cultivation with a series of detector cells. No evidence of infectivity was detected by reverse transcriptase, mink cell S+L- focus assay or by electron microscopic analysis of the inoculated detector cells after at least four passages in culture.

Animals↗
PubMed1991

Endogenous origin of defective retroviruslike particles from a recombinant Chinese hamster ovary cell line.

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. Continuous-flow ultracentrifugation has been used to concentrate extracellular particles from culture fluid of a recombinant CHO cell subclone for molecular characterization. Particles exhibiting reverse transcriptase activity and associated with mammalian C-type retrovirus structural proteins banded in sucrose gradients at a density characteristic of retroviruses. Examination of gradient-purified particles by electron microscopy revealed morphology and size similar to other retroviruses. Double-gradient-purified particles contained RNA which hybridized to probes for murine leukemia virus, and endogenous Chinese hamster intracisternal A-particle elements. DNA sequence analysis of a cDNA clone isolated from purified particles revealed multiple interruptions of the endonuclease reading frame, providing one possible explanation for the noninfectious nature of the observed particles. Sequences present as RNA in purified particles were also present as conserved, repetitive, provirus sequences in genomic DNA of all CHO cell lines examined and in Chinese hamster liver DNA. The observed particles are therefore likely to be the products of endogenous retroviruslike elements present in the germline of Chinese hamsters.

Animals↗
PubMed1991

Defective endogenous retrovirus-like sequences and particles of Chinese hamster ovary cells.

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. To investigate the origin and expression of these particles, retrovirus-like sequences which are actively transcribed in CHO cells have been cloned and characterized. Two families of sequences related to intracisternal A-particle (IAP) genomes of mice and Syrian hamsters were identified in cytoplasmic RNA from CHO cells (CHO IAP family I and family II). None of the four clones which were sequenced exhibited intact gag, pol, or env open reading frames. Only IAP family II sequences were present in purified extracellular particles of CHO cells. Several cDNA sequences related to mammalian C-type retrovirus genomes were isolated and cloned from gradient-purified, extracellular particles of recombinant CHO cells. All were homologous to the conserved endonuclease domain of murine leukemia virus. Nucleotide sequence analysis of the largest cDNA revealed multiple interruptions of the endonuclease encoding reading frame providing one possible explanation for the non-infectious nature of the particles observed in CHO cells. Both types of retrovirus-like sequences identified in purified extracellular particles of CHO cells (CHO IAP family II and C-type) were present as conserved, moderately repetitive sequences in DNA of all CHO cell lines examined, as well as in DNA from a Chinese hamster liver. It is therefore likely that the extracellular retrovirus-like particles of CHO cells are the products of endogenous provirus elements present in the germline of Chinese hamsters.

Animals↗
PubMed1981

Naturally occurring humoral immunity to endogenous xenotropic and amphotropic type-C virus in the mouse.

Natural humoral cytotoxic antibodies from 13- and 18-month-old BALB/c mice showed a virus-specific complement-dependent activity against target cells productively infected with xenotropic, amphotropic or ecotropic type-C viruses. The cytotoxic activity was lowest against ecotropic virus-shedding cells. Serum obtained from mice less than 12 months old had no such reactivity. The cytotoxic reactivity was found to reside solely in the immunoglobulin M fraction which yielded reactivity comparable to the unfractionated sera concerning both titer and relative reactivities to the target cells infected with different type-C viruses. In hyperimmune mouse serum cytotoxic reactivity resided in IgG and IgM fractions. Examination of serum from individual, normal 18-month-old BALB/c mice revealed that 80-90% of them were cytotoxic against virus non-producer mink target cells expressing gp70 or gaggene product. Absorption of sera from 18-month-old normal BALB/c mice with cells shedding Class II or Class III xenotropic virus, amphotropic virus, Rauscher-MuLV, or Class 1 murine leukemia virus indicate a closer amphotropic-FMR viral subtype specificity of the natural cytotoxic immune response as compared to the amphotropic-xenotropic or amphotropic-ecotropic specificity. The incidence and the level of measured humoral cytotoxic activity was sustained in the tumor-bearing animal up to 28 months of age as compared to the background established in the 18-month-old animal. However, in the non-tumor-bearing animal, the incidence and level of cytotoxic reactivity declined rather rapidly with aging. Te sustained cytotoxic reactivity of the serum from old tumored mice might be involved in the progression of the tumor.

Age Factors↗
PubMed1979

Naturally occurring lymphocyte-mediated immunity to endogenous type-C virus in the mouse. Blocking of the lymphocyte reactivity with antisera to the virus.

The natural immune response in mice to their endogenous type-C viruses involves a complex interaction between cellular and humoral immune mechanisms. The virus-specific immune reactivities are a function of age and appear only subsequent to endogenous virus expression. Cellular immune activity was found to reside in a population of lymphocytes that were characterized as natural killer cells based on their absence of theta surface antigens or immunoglobulin or complement receptors. Cellular and humoral virus-specific immune responses co-occur in the same animal and pretreatment of virus-positive target cells with sera from virus-positive aging mice is capable of partially blocking the cytotoxic activity of reactive lymphocytes. The blocking activity of sera from individual mice increases as a function of age and endogenous virus expression and is highly correlated with the virus-specific complement-dependent cytotoxic activity of these sera. Mouse sera, whether naturally immune or immune as a result of hyperimmunization with type-C virus, exhibit blocking activity that can be removed by absorption with purified type-C virus or purified viral glycoprotein (gp 70) but not by absorption with noninfected syngeneic cells. High-titered and highly specific antisera directed against certain individual R-MuLV structural proteins reveal blocking activity. Monospecific antisera to gp 70 and p 12 exhibited high-titered blocking reactivities which are absorbable by the respective purified proteins. Blocking activity of antisera directed against other viral structural proteins could not be excluded with certainty. These findings raise the possibility that immunity in the mouse to endogenous type-C virus or virus-infected cells involves competition between serum-blocking activity and natural-killer cell activity and further provides a unique model system for studying the mechanism of action of blocking antisera known to have monospecific reactivity against defined and purifiable transplantation antigens.

Aging↗
PubMed1978

Cytoplasmic DNA synthesis in rhinovirus type 14-inoculated KB cells.

Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.

Cell Line↗
PubMed1977

Particles resembling oncornaviruses. Spontaneous release from cultured meningioma cells.

A cell culture from thoracic cord meningioma tissue obtained at surgery was maintained for several passages in cell culture. These cells spontaneously released particles with an RNA of high-molecular weight (90S to 95S) and a density similar to that of type C oncornaviruses. The implications of these results and similar findings by others are discussed.

Adolescent↗
PubMed1977

Altered sensitivity of Rous sarcoma virus transformed cells to inhibition of RNA synthesis by alpha-amanitin.

Chick embryo cells transformed by Rous sarcoma virus (RSV) continue to synthesize 40--50 percent of control amounts of RNA following 12--24 hour exposure to 2 microng/ml of the toxin whereas normal chick embryo cells similarly treated synthesize less than 5 percent of control amounts of RNA. Analysis of cells treated with alpha-amanitin, or the resistant forms I and III polymerase, do not increase in infected cells over the levels found in uninfected control cells during the first 24 hours following infection indicating that increase in polymerase levels in infected cells does not account for the observed resistance. No significant difference was detected in the sensitivity to alpha-amanitin in the form II polymerase isolated from normal and transformed cells; The greater sensitivity of normal cells to alpha-amanitin can be reduced by growing the cells at low cell density but the resistance of RSV transformed cells is not significantly altered by changes in cell density. The results suggest that the resistance of RSV transformed cells may be related to altered control of density dependent contact-inhibition of the transformed cells.

Amanitins↗
PubMed1977

A continuous line of Rous sarcoma virus-transformed chick embryo cells.

A continuous line of Rous sarcoma virus (RSV)-transformed chick embryo cells was established. The cells, designated RTAZ-1, which constitute the only known line of continuously growing RSV-transformed cells of chick embryo origin, grow rapidly, display uniform morphology, and perpetually release large amounts of RSV (Rous-associated virus, type 1). RTAZ-1 cells display a heteroploid chromosome complement with 92-94 chromosomes characteristic of chicken cells. Molecular hybridization studies demonstrated that DNA from these cells hybridized with normal chick DNA but not with human (KB) cell DNA, thus providing additional evidence of the chicken origin of RTAZ-1.

Animals↗