The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

Exploring associations between the teat apex metagenome and Staphylococcus aureus intramammary infections in primiparous cows under organic directives.

The primary objective of this study was to identify associations between the prepartum teat apex microbiome and the presence of Staphylococcus aureus intramammary infections (IMI) in primiparous cows during the first 5 weeks after calving. We performed a case-control study using shotgun metagenomics of the teat apex and culture-based milk data collected longitudinally from 710 primiparous cows on five organic dairy farms. Cases had higher odds of having S. aureus metagenomic DNA on the teat apex prior to parturition compared to controls (OR = 38.9, 95% CI: 14.84-102.21). Differential abundance analysis confirmed this association, with cases having a 23.8 higher log fold change (LFC) in the abundance of S. aureus in their samples compared to controls. Of the most prevalent microorganisms in controls, those associated with a lower risk of post-calving S. aureus IMI included Microbacterium phage Min 1 (OR = 0.37, 95% CI: 0.25-0.53), Corynebacterium efficiens (OR = 0.53, 95% CI: 0.30-0.94), Kocuria polaris (OR = 0.54, 95% CI: 0.35-0.82), Micrococcus terreus (OR = 0.64, 95% CI: 0.44-0.93), and Dietzia alimentaria (OR = 0.45, 95% CI: 0.26-0.75). Genes encoding for Microcin B17 AMPs were the most prevalent on the teat apex of cases and controls (99.7% in both groups). The predicted abundance of genes encoding for Microcin B17 was also higher in cases compared to controls (LFC 0.26). IMPORTANCE: Intramammary infections (IMI) caused by Staphylococcus aureus remain an important problem for the dairy industry. The microbiome on the external skin of the teat apex may play a role in mitigating S. aureus IMI risk, in particular the production of antimicrobial peptides (AMPs) by commensal microbes. However, current studies of the teat apex microbiome utilize a 16S approach, which precludes the detection of genomic features such as genes that encode for AMPs. Therefore, further research using a shotgun metagenomic approach is needed to understand what role prepartum teat apex microbiome dynamics play in IMI risk.

Bilateral vertebral arteries entering the C4 foramen transversarium with the left vertebral artery originating from the aortic arch.

Vertebral arteries (VAs) serve as major blood vessels to the central nervous system. VAs typically arise from the subclavian arteries and ascend separately within the transverse foramina of the cervical vertebrae (C6-C1) before entering the skull at the foramen magnum and joining at the base of the pons to form the basilar artery of the vertebrobasilar circulation. Therefore, variations in the origin and anatomic course of the VAs have implications for invasive medical procedures involving the superior thoracic/cervical regions or the cervical vertebrae. The current case report describes variation in the entry point of both VAs and the site of origin of the left vertebral artery. The variation was revealed during routine dissection of a 72-year-old female cadaver. It was found that the left vertebral artery originated directly from the aortic arch to abnormally enter the transverse foramen of C4 instead of the transverse foramen of C6. The right vertebral artery arose as usual from the right subclavian artery. However, the right vertebral artery also directly entered the transverse foramen of C4 instead of the transverse foramen of C6.

Investigating the cow skin and teat canal microbiomes of the bovine udder using different sampling and sequencing approaches.

There is a need for standardized, efficient, and practical sampling methods to support large population-based studies of the internal and external epithelial microbiomes of the bovine udder. The primary objective of this study was to evaluate different sampling devices for the isolation of microbial DNA originating from the internal and external teat epithelium. Secondary objectives were to survey and compare the microbial diversity of external and teat canal epithelial microbiomes using amplicon and shotgun metagenomic sequencing approaches. To address these objectives, we enrolled a convenience sample of 24 Holstein dairy cows and collected samples from the external epithelium at the base of udder, the external teat barrel epithelium, the external teat apex epithelium, and the teat canal epithelium. Extracted DNA was quantified and subjected to PCR amplification of the V4 hypervariable region of the 16S rRNA gene and sequenced on the Illumina MiSeq platform (Illumina Inc., San Diego, CA). A subset of samples was subjected to a shallow shotgun metagenomic assay on the Illumina HiSeq platform. For samples collected from the external teat epithelium, we found that gauze squares consistently yielded more DNA than swabs, and Simpson's reciprocal index of diversity was higher for gauze than for swabs. The teat canal epithelial samples exhibited significantly lower diversity than the external sampling locations, but there were no significant differences in diversity between teat apex, teat barrel, and base of the udder samples. There were, however, differences in the microbial distribution and abundances of specific bacteria across external epithelial surfaces. The proportion of shotgun sequence reads classified as Bos taurus was highly variable between sampling locations, ranging from 0.33% in teat apex samples to 99.91% in teat canal samples. These results indicate that gauze squares should be considered for studying the microbiome of the external epithelium of the bovine udder, particularly if DNA yield must be maximized. Further, the relative proportion of host to non-host DNA present in samples collected from the internal and external teat epithelium should be considered when designing studies that utilize shotgun metagenomic sequencing.

The addition of ticarcillin-clavulanic acid to INRA 96 extender for stallion semen cooling.

REASONS FOR PERFORMING STUDY: A commonly used commercial extender (i.e. INRA 96) contains antimicrobials that may have limited effectiveness. Therefore, addition of ticarcillin-clavulanic acid to this extender is a widespread procedure in the equine breeding industry in the United States. However, such practice has not been critically evaluated. OBJECTIVES: To evaluate the addition of ticarcillin-clavulanic acid to INRA 96 and different extender and antimicrobial storage conditions on sperm function and antimicrobial effectiveness. METHODS: Gel-free semen (42 ejaculates from 14 mature Quarter Horse stallions) was extended with INRA 96 and stored for 24 h in an Equitainer II. The effects of added ticarcillin-clavulanic acid and different extender storage procedures on sperm motion characteristics (by computer-assisted analysis), sperm membrane integrity (by fluorescence-based measurement) and suppression of bacterial growth (by aerobic and anaerobic culture methods) were evaluated using analysis-of-variance and Chi-square statistical methods. The P value for significance was set at < 0.05. RESULTS: Freezing and thawing of modified or unmodified extender prior to use for stallion semen resulted in reduced sperm quality post cooling for 24 h, as evidenced by a significant reduction in sperm motility (i.e. total and progressive) and sperm membrane integrity. Addition of ticarcillin-clavulanic acid to extender resulted in higher sperm velocity when the reconstituted antimicrobial was subjected to cooled storage, as compared with frozen storage, prior to use. Only 28 of 42 ejaculates (67%) yielded presence of bacteria in neat semen but addition of ticarcillin-clavulanic acid to INRA 96 was not different than INRA 96 alone for inhibiting growth of bacteria (98 vs. 94%, respectively). CONCLUSIONS: Addition of ticarcillin-clavulanic acid (1 mg/ml) to INRA 96 did not adversely affect sperm quality in extended semen after cooled storage. Extender freezing and thawing prior to use had detrimental effects on sperm quality. POTENTIAL RELEVANCE: These data suggest that INRA 96 should not be frozen and thawed prior to use. Addition of ticarcillin-clavulanic acid to INRA 96 did not impair sperm quality. All extender treatments effectively controlled the bacterial growth compared with neat semen.

An evaluation of four CT-MRI co-registration techniques for radiotherapy treatment planning of prone rectal cancer patients.

OBJECTIVES: MRI is the preferred staging modality for rectal carcinoma patients. This work assesses the CT-MRI co-registration accuracy of four commercial rigid-body techniques for external beam radiotherapy treatment planning for patients treated in the prone position without fiducial markers. METHODS: 17 patients with biopsy-proven rectal carcinoma were scanned with CT and MRI in the prone position without the use of fiducial markers. A reference co-registration was performed by consensus of a radiologist and two physicists. This was compared with two automated and two manual techniques on two separate treatment planning systems. Accuracy and reproducibility were analysed using a measure of target registration error (TRE) that was based on the average distance of the mis-registration between vertices of the clinically relevant gross tumour volume as delineated on the CT image. RESULTS: An automated technique achieved the greatest accuracy, with a TRE of 2.3 mm. Both automated techniques demonstrated perfect reproducibility and were significantly faster than their manual counterparts. There was a significant difference in TRE between registrations performed on the two planning systems, but there were no significant differences between the manual and automated techniques. CONCLUSION: For patients with rectal cancer, MRI acquired in the prone treatment position without fiducial markers can be accurately registered with planning CT. An automated registration technique offered a fast and accurate solution with associated uncertainties within acceptable treatment planning limits.

Measurement of cone beam CT coincidence with megavoltage isocentre and image sharpness using the QUASAR Penta-Guide phantom.

For image-guided radiotherapy (IGRT) systems based on cone beam CT (CBCT) integrated into a linear accelerator, the reproducible alignment of imager to x-ray source is critical to the registration of both the x-ray-volumetric image with the megavoltage (MV) beam isocentre and image sharpness. An enhanced method of determining the CBCT to MV isocentre alignment using the QUASAR Penta-Guide phantom was developed which improved both precision and accuracy. This was benchmarked against our existing method which used software and a ball-bearing (BB) phantom provided by Elekta. Additionally, a method of measuring an image sharpness metric (MTF(50)) from the edge response function of a spherical air cavity within the Penta-Guide phantom was developed and its sensitivity was tested by simulating misalignments of the kV imager. Reproducibility testing of the enhanced Penta-Guide method demonstrated a systematic error of <0.2 mm when compared to the BB method with near equivalent random error (s=0.15 mm). The mean MTF(50) for five measurements was 0.278+/-0.004 lp mm(-1) with no applied misalignment. Simulated misalignments exhibited a clear peak in the MTF(50) enabling misalignments greater than 0.4 mm to be detected. The Penta-Guide phantom can be used to precisely measure CBCT-MV coincidence and image sharpness on CBCT-IGRT systems.

Intracrystalline proteins and the hidden ultrastructure of calcium oxalate urinary crystals: implications for kidney stone formation.

The external appearance of urinary calcium oxalate (CaOx) crystals suggests that they are solid, homogeneous structures, despite their known association with proteins. Our aim was to determine whether proteins comprising the organic matrix of CaOx crystals are superficial or intracrystalline in order to clarify the role of urinary proteins in the formation of kidney stones. CaOx crystals were precipitated from centrifuged and filtered, or ultrafiltered, healthy human urine. They were then treated with dilute NaOH to remove bound proteins, partially demineralized with EDTA, or fractured and subjected to limited proteolysis before examination by low-resolution scanning electron microscopy or field emission scanning electron microscopy. Crystals precipitated from centrifuged and filtered urine had a complex interior network of protein distributed throughout the mineral phase, which appeared to comprise closely packed subcrystalline particles stacked in an orderly array among an amorphous organic matrix. This ultrastructure was not evident in crystals deposited in the absence of macromolecules, which were completely solid. This is the first direct evidence that crystals generated from cell-free systems contain significant amounts of protein distributed throughout a complex internal cribriform ultrastructure. Combined with mineral erosion in the acidic lysosomal environment, proteins inside CaOx crystals would render them susceptible to attack by urinary and intracellular renal proteases and facilitate their further dissolution or disruption into small particles and ions for removal by exocytosis. The findings also have broader ramifications for industry and the materials sciences, as well as the development and resorption of crystals in biomineralization systems throughout nature.

The hole truth: intracrystalline proteins and calcium oxalate kidney stones.

The ultimate aim of our research is to understand the role of macromolecules in the formation of human kidney stones, particularly their interactions with calcium oxalate (CaOx) crystals. The invariable association of stones with proteins raises the possibility that proteins play a role in their formation, similar to the role of proteins in healthy biomineralization. Do these proteins induce mineralization? Are they merely a response to the disease process? Or are they protective molecules that were overwhelmed by mineral supersaturation? A protein of particular interest is fragment 1 (F1) of prothrombin. We have shown that mRNA for prothrombin is present in the kidney. Because the F1 fragment of prothrombin present in urine is slightly different from that found in the blood, we refer to this protein as "urinary prothrombin fragment 1" (UPTF1). Available evidence suggests that the kidney manufactures the protein for protection against stone disease and that the protein has a directive role in stone formation. We now have evidence that proteins are interred within CaOx crystals precipitated from human urine, where it is distributed in continuous channels. These proteins could facilitate crystal deconstruction and removal after attachment to the renal epithelium and endocytosis. We suspect that the formation of CaOx crystals in the urine is a normal process designed to permit harmless disposal of an excess of calcium, oxalate, or both. The incorporation of proteins provides a second line of defense against stone formation by enabling the destruction and removal of retained crystals. Understanding the basic molecular strategies by which plants produce protein-containing CaOx crystals may provide insight into human CaOx stone formation.

Characterization of monoclonal antibody CIBCNSH3 generated to the human EGF receptor.

Monoclonal antibody CIBCNSH3 of IgG1 isotype has been generated against human epidermal growth factor receptor (EGFR) using MDA MB 468 breast carcinoma cell line as immunogen. Earlier studies have revealed that this MAb blocked growth factor-receptor interaction and thus inhibited cell proliferation and tumor growth. In the present paper, this MAb has been extensively characterized to evaluate its application in the study of human cancers. The results were compared with those obtained using a control MAb ICR 62 specific to EGFR. Competitive assay showed that this MAb bound to an epitope in the extracellular domain of the EGFR to which MAb ICR 62 also bound. This MAb immunoprecipitated the 170 kD glycoprotein. The specificity was further confirmed by the formation of a single discrete band in western blot analysis. By flow cytometric analysis this monoclonal antibody revealed high binding affinity with MDA MB 468 cells. By immunocytochemical assay, out of 35 breast tumors studied, 40% were found to exhibit strong cell membrane staining and in the case of 25 oral cancers studied, 56% were strong positive. High expression of EGFR was observed in MDA MB 468 cells and HN 5 cells. These studies clearly indicate that MAb CIBCNSH3 might prove useful to identify tumors with high level of expression of EGFR associated with poor prognosis.

Anti-tumor activity of monoclonal antibody CIBCNSH3 generated to the human EGF receptor.

The overexpression of the human epidermal growth factor receptor (EGFR) has been demonstrated in many malignancies like squamous cell carcinoma of the head and neck, cervix, breast etc. which are most prevalent in India. This is often associated with poor prognosis and high mortality in these patients. Monoclonal antibodies generated against EGFR which inhibit binding of ligands like EGF to their receptor have anti-tumor activity and hence therapeutic application. One such monoclonal antibody designated as CIBCNSH3 generated in our laboratory has been found to recognize an epitope in the extracellular domain of EGFR by immunoprecipitation. By immunoperoxidase test this antibody exhibited strong reactivity to EGFR in head and neck cancers and breast cancers studied. It also inhibited the binding of Epidermal Growth Factor (EGF) to its receptor on MDA MB468 breast cancer cells rich in EGFR as revealed by competitive binding assay using 125I EGF, indicating its anti-tumor activity. The in vivo therapeutic efficacy has been demonstrated by injecting i.p. into tumor bearing mice 200 micrograms of the antibody for 4 consecutive days and then 100 micrograms twice a week resulting in complete regression of tumors of initial tumor size of 0.5-1.0 cm diameter. These results were compared with a control antibody against EGFR and also a nonspecific antibody which were administered to different groups of animals. In vivo studies performed using cell lines in culture like MDA MB468, MDA MB157 and HN5 with overexpression of EGFR revealed 98% cell death when incubated with different concentrations of the antibody. This monoclonal antibody seems to have a promising future application as therapeutic agent for tumors which overexpress EGFR.

Brk, a breast tumor-derived non-receptor protein-tyrosine kinase, sensitizes mammary epithelial cells to epidermal growth factor.

brk (breast tumor kinase) shows homology to the src family of non-receptor protein-tyrosine kinases and is expressed in breast carcinomas. In order to investigate the role of brk in breast tumor development, we have examined the growth and transformation properties of human mammary epithelial cells engineered to overexpress Brk. Interestingly, like c-Src, overexpression of Brk leads to sensitization to EGF, and also results in a partially transformed phenotype. Further investigation of the latter activity was attempted by mutational analysis, targeting key residues known to affect tyrosine kinase activity in Src-like kinases. Mutation of amino acid residue Lys-219 to Met, by analogy to Src, abolished both kinase activity and transformation capacity. Mutation of amino acid residue Tyr-447 to Phe, however, resulted in a decrease in transforming potential without affecting kinase activity. These results suggest that while Src and Brk share some functional properties, they act differently during transformation. These differences are discussed in the context of the mechanisms underlying breast cancer development.

Radioimmunotherapy of breast cancer xenografts with monoclonal antibody ICR12 against c-erbB2 p185: comparison of iodogen and N-succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate radioiodination methods.

C-erbB2 p185 is a proto-oncogene product expressed in 25-30% of human invasive breast cancers that is associated with poor prognosis and resistance to endocrine therapy and chemotherapy. It is minimally expressed in normal adult tissues (M. F. Press et al., Oncogene, 5: 953-962, 1990). For this reason, it is an attractive target for radioimmunotherapy and other antibody-directed therapies. ICR12 is a rat IgG2a monoclonal antibody directed against a protein epitope of the external domain of the c-erbB2 p185. We performed experiments to optimize the direct iodination of ICR12 with 131I using the IodoGen method, and we found impairment of immunoreactive fraction with increasing specific activity. N-Succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate (MATE) is a tin ester that can be radioiodinated easily and then coupled to the epsilon-amino group of lysine residues. This method has been shown to have improved uptake in tumors compared with antibody labeled by direct iodination (P. K. Garg et al., Nucl. Med. Biol., 20: 379-387, 1993). ICR12 could be labeled up to 16 mCi/mg by this technique without loss of immunoreactive fraction. Whole-body retention of MATE-labeled ICR12 was less than IodoGen (P < 0.0001). Radioimmunotherapy experiments in athymic mice bearing established MDA MB 361 human breast cancer xenografts showed growth inhibition for > 24 days at a dose of 600 microCi/mouse (P < 0.0001) when labeled by the IodoGen technique, and 12 days using the MATE method (P < 0.0001).

A monoclonal antibody that recognizes alkali-stabilized melphalan-DNA adducts and its application in immunofluorescence microscopy.

Monoclonal antibodies were produced that recognized alkali-stabilized modifications of DNA formed by the anticancer drug melphalan in order to permit measurement of melphalan-DNA adducts in individual cells by immunofluorescent staining. Antibody Amp4/42 did not bind to alkali-treated control DNA or to DNA that had been alkylated with melphalan but not exposed to alkali. In a competitive enzyme-linked immunoadsorbent assay using DNA that had been reacted with radioactive melphalan in simple solution a 50% reduction in assay signal was caused by approximately 100 fmol total melphalan-DNA adducts/assay well. This sensitivity was only slightly influenced by heat denaturation of the DNA before alkylation or by the frequency of alkylated sites on DNA. The heat stability of the adducts recognized by Amp4/42 was greatly increased by the alkali-induced change which, in 0.1 M NaOH at 37 degrees C, was complete by 30 min. Amp4/42 appears to recognize a ring-opened structure resulting from alkaline hydrolysis of 7-alkyldeoxyguanosine. Melphalan-DNA adducts formed in mammalian cells showed an alkali-induced increase in immunoreactivity which occurred at a similar rate to that seen in DNA that had been alkylated in simple solution, but their maximum overall immunoreactivity was approximately 10-fold lower. This indicated that in cells the adducts recognized by Amp4/42 were formed or persisted as a smaller proportion of total adducts compared with alkylation of pre-purified DNA in simple solution. This antibody permitted immunofluorescent detection of melphalan-DNA adducts in single cells.

Regression of established breast carcinoma xenografts with antibody-directed enzyme prodrug therapy against c-erbB2 p185.

The enzyme carboxypeptidase G2 (CPG2) was conjugated to the rat IgG2a monoclonal antibody (mAb) ICR12, which recognizes the external domain of the human c-erbB2 protooncogene product. The conjugate retained antigen-binding and enzyme activity. Radiolabeled conjugate localized efficiently and stably to MDA MB 361 breast carcinoma xenografts, which overexpress the c-erbB2 gene product p185. Radiotracer determinations and plasma enzyme activity studies in nu/nu mice gave conjugate blood clearance rate half-lives of approximately 4 days. In separate antibody-directed enzyme prodrug therapy regimes, one dose of the 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid prodrug was administered to nu/nu mice bearing established MDA MB 361 tumors (mean volume, 170-260 mm3). In mice which had received ICR12-CPG2 12-14 days previously, sustained dose-dependent tumor stasis or regressions were effected, which in some cases persisted throughout observation periods of up to 90 days. In control mice which had received the isotype-matched irrelevant mAb ICR16-CPG2 conjugate, tumors grew progressively, as did those in mice treated with prodrug alone, or treated simultaneously with ICR12-CPG2 and prodrug at the maximum tolerated dose. Control chemotherapy with conventional drugs proved toxic and induced only minimal growth delays.

Presentation of anti-idiotypic antibody is sensitive to ionizing radiation.

We have been investigating the generation of specific immune responses using monoclonal anti-idiotypic Abs (Ab2) as surrogate tumor Ag. We have prepared a series of idiotypic mAbs (Ab1) from CBH/cbi rats bearing the syngeneic sarcoma HSN and have used these Ab1 to generate autologous Ab2. By using the autologous Ab2 as Ag, we have isolated T cell lines from CBH/cbi rats that proliferate specifically in the presence of the Ab2, with spleen cells as APC. Specific proliferation of the T cells was prevented if the spleen cells used for presentation were irradiated with conventional doses of x-rays (1000 rad) just before use. Titration of the radiation response showed that the capacity of the spleen cells to present Ag decreased exponentially with x-ray doses of up to 100 rad, at which dose presentation was virtually abolished. The same irradiated spleen cells were fully competent to present OVA to CBH/cbi-derived rat T cell lines specific for this Ag. Preincubating the APC with Ag before irradiation abrogated the effect of x-irradiation on the presentation of Ab2. We conclude that, in this rat system, the presentation of autologous Ab2 is highly sensitive to the effects of low doses of x-rays. The clinical significance of these findings is discussed.

Preclinical models for the evaluation of targeted therapies of metastatic disease.

It has been estimated that approx 60-70% of cancer patients harbor overt or subclinical metastases at diagnosis, and it is the eradication of such systemic disease that largely determines survival. Preclinical tumor model systems employed to evaluate potential new treatment strategies should aim to represent the process and patterns of metastasis of their clinical counterparts as closely as possible. Severe combined immune-deficient (SCID) and nu/nu mice have been extensively used as hosts for the growth of human tumor cell lines and in some cases fresh tumor material. However, in most instances the resulting neoplasms fail to metastasize, and the aberrant immune systems of such animals has limited their use mainly to passive therapies of localized disease. Recently, the development of specially selected tumor variants and the use of appropriate orthotopic sites for implantation has provided several models in which dissemination can be demonstrated. Where the gene coding for a potential target antigen has been cloned, and where its overexpression or mutation is associated with malignancy (e.g., c-erbB-2, H-ras), transgenic mice may yield tumors that will develop in these immunocompetent hosts. In some cases such tumors exhibit metastasis. A third approach is to transfect human genes of interest into appropriate rodent tumors expressing the desired metastatic phenotype. These various approaches are compared with particular reference to mammary carcinoma biology.