Review Canine Tuberculosis - An Emerging Concern.

Canine mycobacterial disease was first recognised over 100 years ago but is now an emerging concern. All reported cases of tuberculous disease in dogs have been caused by infection with one of three Mycobacterium tuberculosis-complex (MTBC) organisms (M. tuberculosis, Mycobacterium bovis, and Mycobacterium microti). Molecular PCR and interferon-gamma release assays offer alternative or complementary diagnostic pathways to that of specialist culture, which is limited by availability, sensitivity, and the time it takes to get a result. Optimised triple antimicrobial protocols offer an excellent chance of a successful outcome in dogs where treatment can be considered and is attempted. In this review, the clinical presentation, diagnosis, treatment, and prognosis of canine tuberculosis are discussed.

Hepatic Gene Expression of Angiogenic and Regeneration Markers in Cats with Congenital Portosystemic Shunts (CPSS).

Congenital portosystemic shunts (CPSS) are vascular anomalies resulting in liver hypoplasia and hepatic insufficiency. Cats with CPSS typically show signs of hepatic encephalopathy associated with increased ammonia, inflammatory cytokines, and oxidative stress. Surgical attenuation of the CPSS results in improved liver function, resolution of clinical signs, and increased portal blood flow. Hepatic gene expression has not previously been investigated in cats with CPSS. Here, we compared the hepatic expression of genes involved in the urea cycle (CPS1, NAGS), angiogenesis (VEGFR2, NPPA, NPR1, NPPC, NPR2, HIF1a), liver regeneration (SERPINB1, HGF, TGFß), and metabolism (FGF21) from a small series of cats (n = 18) with CPSS to that of control cats (n = 10). The expression of TGFß, VEGFR2, HGF, FGF21, and CPS1 was significantly elevated in liver biopsies from cats with CPSS. Cats that could only tolerate partial closure of their CPSS had increased hepatic expression of SERPINB1, HIF1a, and NPR2 compared with those that could tolerate complete ligation. Furthermore, there were no significant correlations between gene expression and pre-operative plasma ammonia concentrations in cats with CPSS. The changes in hepatic gene expression in cats with CPSS are in direct contrast to those seen in dogs with CPSS, suggesting alternative mechanisms may be involved in mediating hepatic changes in cats with CPSS.

Canine tuberculosis: A review of 18 new and 565 previously reported confirmed cases.

Cases of canine tuberculosis, a zoonotic infection of significant public health significance, are typically only sporadically reported in the literature. For this observational study, case details were collated both retrospectively and prospectively for dogs infected with Mycobacterium tuberculosis-complex (MTBC) organisms. A total of 18 previously unreported cases as well as 565 historically reported confirmed cases were reviewed. A variety of diagnostic techniques were used to make a confirmed diagnosis of tuberculosis (culture, interferon-gamma release assay [IGRA], and PCR). The reference standard for diagnosis is culture; however, this was negative or not attempted in some dogs. Where fully speciated, all cases were caused by infection with one of three MTBC organisms: M. tuberculosis, Mycobacterium bovis, or Mycobacterium microti. This study includes the first documented canine infections with M. microti in the UK. All cases were assigned to one of four clinical groups based on the presenting signs: 44.1% were primarily pulmonary, 14.5% were primarily abdominal, and the remainder were disseminated or miscellaneous. The development of adjunctive tests remains necessary to support early treatment decisions pending reporting of culture for MTBC organisms, which can take weeks to months. Definitive treatment, where attempted, was successful in most cases. Of the 13 dogs treated by the authors with triple combination antimicrobial therapy, a good clinical outcome was seen in 12 (92%) of them.

Retrospective study and outcome of 307 cats with feline infectious peritonitis treated with legally sourced veterinary compounded preparations of remdesivir and GS-441524 (2020-2022).

OBJECTIVES: Feline infectious peritonitis (FIP) is a serious disease that arises due to feline coronavirus infection. The nucleoside analogues remdesivir and GS-441524 can be effective in its treatment, but most studies have used unregulated products of unknown composition. The aim of the present study was to describe the treatment of FIP using legally sourced veterinary-prescribed regulated veterinary compounded products containing known amounts of remdesivir (injectable) or GS-441524 (oral tablets). METHODS: Cats were recruited via email advice services, product sales contacts and study publicity. Cats were excluded if they were deemed unlikely to have FIP, were not treated exclusively with the veterinary compounded products, or if there was a lack of cat and/or treatment (including response) data. Extensive cat and treatment data were collected. RESULTS: Among the 307 cats recruited, the predominant type of FIP was most commonly abdominal effusive (49.5%) and then neurological (14.3%). Three treatment protocols were used; remdesivir alone (33.9%), remdesivir followed by GS-441524 (55.7%) and GS-441524 alone (10.4%). The median (range) initial treatment period duration and longest follow-up time point after starting treatment were 84 (1-330) days and 248 (1-814) days, respectively. The most common side effect was injection pain (in 47.8% of those given subcutaneous remdesivir). Of the 307 cats, 33 (10.8%) relapsed, 15 (45.5%) during and 18 (54.5%) after the initial treatment period. At the longest follow-up time point after completion of the initial treatment period, 84.4% of cats were alive. The cats achieving a complete response within 30 days of starting treatment were significantly more likely to be alive at the end of the initial treatment period than those cats that did not. CONCLUSIONS AND RELEVANCE: Legally sourced remdesivir and GS-441524 products, either alone or used sequentially, were very effective in the treatment of FIP in this group of cats. Variable protocols precluded statistical comparison of treatment regimens.

Effect of an antimicrobial stewardship intervention on the prescribing behaviours of companion animal veterinarians: A pre-post study.

BACKGROUND: Antimicrobial stewardship is a cornerstone of efforts to combat antimicrobial resistance. We evaluated the impact of a formal discussion of antimicrobial stewardship for dogs and cats on systemic antimicrobial prescribing behaviours among companion animal veterinarians. METHODS: Electronic health records including information about the prescription of antimicrobials were collected from a multisite UK veterinary practice between 2017 and 2020. We undertook interrupted time series analysis using a quasi-Poisson model to compare the pre- and postintervention change in level and slope for multiple outcomes. RESULTS: After the intervention, there were sustained reductions in the prescription rate of cefovecin to cats and metronidazole to dogs and increases in amoxicillin-clavulanic acid prescribing. There was no evidence for an immediate change in overall prescribing rates in either species, although rates increased over the 12 months after the intervention. There was a transient increase in the proportion of dogs who had their weight recorded at the time of prescription. Results suggest decreases in the prescription of off-licence antimicrobials in both species and the likelihood of underdosing in dogs. CONCLUSIONS: Discussion of antimicrobial stewardship is more likely to influence the antimicrobial choice than whether to prescribe or not. Interventions may benefit by focusing on appropriate antimicrobial selection rather than overall prescription frequency.

Subarachnoid diverticulum associated with feline infectious peritonitis in a Siberian cat.

CASE SUMMARY: A 7-month-old Siberian cat was presented for investigation of acute onset multifocal neurological deficits. Neurological examination documented dull mental status and an ambulatory left hemiparesis. Serum biochemistry documented marked hyperglobulinaemia. MRI of the brain identified marked leptomeningeal contrast enhancement extending along the brainstem caudally to involve the cranial cervical spinal cord. MRI of the cervical spine further identified a subarachnoid diverticulum that extended from the level of the obex to the C2-C3 vertebrae. Cerebrospinal fluid quantitative RT-PCR was positive for the presence of feline coronavirus. Histopathology revealed pyogranulomatous meningitis and choroid plexitis, uveitis and nephritis. RELEVANCE AND NOVEL INFORMATION: This article describes the first reported case of a subarachnoid diverticulum associated with feline infectious peritonitis.

Colony Stimulating Factors in Early Feline Infectious Peritonitis Virus Infection of Monocytes and in End Stage Feline Infectious Peritonitis; A Combined In Vivo And In Vitro Approach.

Feline coronavirus (FCoV) infection initiates monocyte-associated viremia and viral persistence. Virus-infected, -activated monocytes also trigger feline infectious peritonitis (FIP), a fatal systemic disease of felids typified by granulomatous (peri)phlebitis. Currently, the exact mechanisms inducing monocyte activation and FIP are unknown. This study attempted to identify the potential immediate effect of virulent FCoV on colony-stimulating factor (CSF) (granulocyte (G)-CSF, monocyte (M)-CSF and granulocyte-monocyte (GM)-CSF levels through in vitro assessment, alongside prototypical pro- and anti-inflammatory mediators (interleukin (IL)-1, IL-6, IL-12p40, tumor necrosis factor (TNF)-α, and IL-10); this was assessed alongside the in vivo situation in the hemolymphatic tissues of cats euthanized with natural end-stage FIP. For the in vitro work, isolated monocytes from SPF cats were cultured short-term and infected with the FIP virus (FIPV) strain DF2. Mediator transcription was assessed by quantitative reverse transcriptase PCR (RT-qPCR) at 3, 6 and 9 h post infection (hpi), and in the post-mortem samples of bone marrow, spleen, and mesenteric lymph nodes (MLN) of cats with FIP. We observed limited and transient changes in cytokine transcription in monocytes after infection, i.e., a significant increase of IL-6 at 3 hpi and of GM-CSF over the 3 and 6 hpi period, whereas M-CSF was significantly decreased at 9 hpi, with a limited effect of age. The findings indicate that the infection induces expansion of the monocyte/macrophage population, which would ensure the sufficient supply of cells for consistent viral replication. In natural disease, the only upregulation was of G-CSF in the MLN, suggesting either immune exhaustion or an active downregulation by the host as part of its viral response.

Evaluation of Interferon-Gamma Polymorphisms as a Risk Factor in Feline Infectious Peritonitis Development in Non-Pedigree Cats-A Large Cohort Study.

Feline infectious peritonitis (FIP) is a common infectious cause of death in cats, with heritable host factors associated with altered risk of disease. To assess the role of feline interferon-gamma gene (fIFNG) variants in this risk, the allele frequencies of two single nucleotide polymorphisms (SNPs) (g.401 and g.408) were determined for non-pedigree cats either with confirmed FIP (n = 59) or from the general population (cats enrolled in a large lifetime longitudinal study; n = 264). DNA was extracted from buccal swabs or tissue samples. A pyrosequencing assay to characterize the fIFNG SNPs was designed, optimized and subsequently performed on all samples. Genotype and allele frequency were calculated for each population. Characterization of the target SNPs was possible for 56 of the cats with FIP and 263 of the cats from the general population. The SNPs were in complete linkage disequilibrium with each other. There was an association between FIP status and genotype (χ2; p = 0.028), with a reduced risk of developing FIP (χ2; p = 0.0077) associated with the genotype TT at both positions. These results indicate that, although fIFNG variants may be associated with altered risk of disease, the prevalence of individual variants within both populations limits application of their characterization to breeding purposes.

The Effect of Natural Feline Coronavirus Infection on the Host Immune Response: A Whole-Transcriptome Analysis of the Mesenteric Lymph Nodes in Cats with and without Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a coronavirus-induced disease of cats, in which the immune system is known to play a crucial, but complex, role in the pathogenesis. This role is still incompletely understood, with involvement of both host and viral factors. To evaluate differential gene expression and pathway involvement in feline coronavirus (FCoV) infection and FIP, we applied next-generation RNA-sequencing of the mesenteric lymph nodes from cats with naturally-acquired FIP, as well as those with systemic FCoV infection without FIP, and those with neither. Viral infection was associated with upregulation of viral defenses regardless of the disease state, but to a greater degree in FIP. FIP was associated with higher pro-inflammatory pathway enrichment, whilst non-FIP FCoV-positive cats showed lower enrichment of humoral immunity pathways, below that of uninfected cats in the case of immunoglobulin production pathways. This host response is presumed to be protective. In FIP, downregulation of T cell-related processes was observed, which did not occur in non-FIP FCoV-positive cats. These results emphasize the importance of the host's immune balance in determining the outcome of the FCoV infection.

Evaluation of polymorphisms in inflammatory mediator and cellular adhesion genes as risk factors for feline infectious peritonitis.

OBJECTIVES: Feline infectious peritonitis (FIP) is a high mortality infectious disease. Single nucleotide polymorphisms (SNPs) in the genes encoding interferon gamma (IFNG), tumour necrosis factor alpha (TNFA) and dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN; CD209) have been associated with increased and decreased risk of developing FIP. This study was designed to determine whether these associations were present in a UK population of pedigree cats using samples from cats euthanased with a confirmed diagnosis (FIP, n = 22; non-FIP, n = 10) or clinically healthy cats over 11 years of age (n = 3). METHODS: DNA was extracted from tissue (n = 32) or blood (n = 3) and PCR performed for regions of IFNG, TNFA and CD209. PCR amplicons were sequenced, each SNP genotype was determined, and genotype/allele frequency for each SNP and FIP status were compared. RESULTS: No significant association was found between the genotype and FIP status for any SNP analysed. There was a trend for the heterozygous CT genotype at both IFNG g.401 and IFNG g.408 to be associated with FIP (P = 0.13), but this genotype was also found in a substantial proportion of non-FIP cats. There was also a trend for the heterozygous CT genotype at IFNG g.428 to be associated with FIP (P = 0.06), although most cats with FIP had the CC genotype at this locus. No associations were found between any allele at TNFA g.-421, CD209 g.1900, CD209 g.2276, CD209 g.2392 and CD209 g.2713 and FIP. CONCLUSIONS AND RELEVANCE: The use of the IFNG, TNFA and CD209 SNPs described to predict the risk of FIP cannot currently be recommended.

Hyperplastic and fibrosing gastropathy resembling Ménétrier disease in a cat.

CASE SUMMARY: A 3.5-year-old domestic shorthair cat presented with a 6 month history of weight loss and polyphagia. Clinical examination revealed a markedly reduced body condition score (2/9) and a quiet demeanour. Laboratory abnormalities comprised a mild non-regenerative anaemia, stress leukogram, hypoproteinaemia due to hypoalbuminaemia, azotaemia, hypokalaemia, total hypocalcaemia and sub-maximally concentrated urine (specific gravity 1.020). Abdominal ultrasonography revealed marked thickening of the gastric mucosa within the fundus, body and pylorus; the most dorsal portion of the fundus was spared. The thickened mucosa contained multiple small, anechoic cyst-like structures. The gastric submucosa, muscularis and serosa appeared normal. Histopathology, performed on a full-thickness gastric biopsy, revealed mucosal hypertrophy and markedly dilated gastric glands in areas; not all gastric glands were affected, with some appearing normal or atrophic. Focal interstitial fibrosis was present in some areas. The findings of hypoproteinaemia, gastric ultrasonographic changes and histopathology results share several similarities to those reported with Ménétrier disease. RELEVANCE AND NOVEL INFORMATION: Ménétrier disease is a rare condition of the stomach in humans. A similar condition, giant hypertrophic gastritis (or Ménétrier-like disease), has also been described rarely in dogs. To our knowledge, Ménétrier-like disease has not been previously described cats. This case shares features of Ménétrier-like disease, raising the suspicion of a similar aetiopathogenesis.

Update on Feline Hemoplasmosis.

"The wall-less, hemotropic, mycoplasma species Mycoplasma haemofelis, "Candidatus Mycoplasma turicensis" and, to a lesser extent, "Candidatus Mycoplasma haemominutum" have the potential to induce clinical hemolytic anemia in infected cats. Prevalence varies markedly between infecting species, complicated by a chronic carrier state. Accurate and prompt confirmation of infection and identification of the infecting hemoplasma species enables appropriate antibiotics (eg, tetracycline; fluoroquinolone) to be prescribed. Although cats with hemoplasmosis respond rapidly to antibiosis and supportive care, initial monotherapy treatment rarely results in clearance of infection. A protocol now exists for the clearance of the most pathogenic feline hemoplasma M haemofelis."

In vitro and in vivo anticancer activity of 2-acetyl-benzylamine isolated from Adhatoda vasica L. leaves.

One of the important aims of drug discovery for cancer is to find therapeutic agents from natural products that are effective and safe for cancer treatment. In the current study, an alkaloid, 2-acetyl-benzylamine, isolated from Adhatoda vasica, was screened for potent anticancer properties against leukemia cells. We used seven different types of leukemia cells such as CEM, NB-4, MOLM-14, Jurkat, IM-9, K562 and HL-60 for cytotoxic studies. 2-acetyl-benzylamine showed significant cytotoxic properties against MOLM-14 and NB-4 cells with IC50 values of 0.40 and 0.39mM at 24h when compared to other tested cells, respectively. Apoptosis was confirmed by annexin V-FITC/PI kit using flow cytometry and confocal microscope in MOLM-14 and NB-4 cells. In addition, 2-acetyl-benzylamine induced cell cycle arrest at G2/M phase in MOLM-14 cells and G0/G1 phase in NB-4 cells. Apoptosis mechanism was confirmed by RT-PCR and Western blot analysis. Treatment with 2-acetyl-benzylamine decreased the Bcl-2 activity and increased the Bax expression; cytochrome c was released and caspases-3 was activated in MOLM-14 and NB-4 cells. Besides, 2-acetyl-benzylamine inhibited the expression of JAK2/STAT3 in MOLM-14 and NB-4 cells. In vivo administration of 2-acetyl-benzylamine inhibited the growth of MOLM-14 cells in xenograft mice model. Molecular docking study has been performed to investigate the binding mode and to estimate the binding energy of 2-acetyl-benzylamine with the active site of JAK-2, AKT1, FLT3 and Bcl-2. The above findings proved that 2-acetyl-benzylamine could be developed as a potential therapeutic agent against cancer.

In vitro antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone induced apoptosis against COLO320 cells through cytochrome c release caspase mediated pathway with PI3K/AKT and COX-2 inhibition.

The present study investigated the anticancer activity of 2,3-dihydroxy-9,10-anthraquinone against different cancer cells such as MCF-7, COLO320, HepG-2, Skov-3, MOLM-14, NB-4, CEM, K562, Jurkat, HL-60, U937, IM-9 and Vero. 2,3-dihydroxy-9,10-anthraquinone showed good antiproliferative activity against COLO320 cells when compared to other tested cells. The cytotoxicity results showed 79.8% activity at the dose of 2.07 µM with IC50 value of 0.13 µM at 24 h in COLO320 cells. So we chose COLO320 cells for further anticancer studies. mRNA expression was confirmed by qPCR analysis using SYBR green method. Treatment with 2,3-dihydroxy-9,10-anthraquinone was found to trigger intrinsic apoptotic pathway as indicated by down regulation of Bcl-2, Bcl-xl; up regulation of Bim, Bax, Bad; release of cytochrome c and pro-caspases cleaving to caspases. Furthermore, 2,3-dihydroxy-9,10-anthraquinone stopped at G0/G1 phase with modulation in protein levels of cyclins. On the other hand PI3K/AKT signaling plays an important role in cell metabolism. We found that 2,3-dihydroxy-9,10-anthraquinone inhibits PI3K/AKT activity after treatment. Also, COX-2 enzyme plays a major role in colorectal cancer. Our results showed that the treatment significantly reduced COX-2 enzyme in COLO320 cells. These results indicated antiproliferative activity of 2,3-dihydroxy-9,10-anthraquinone involving apoptotic pathways, mitochondrial functions, cell cycle checkpoint and controlling the over expression genes during the colorectal cancer. Molecular docking studies showed that the compound bound stably to the active sites of Bcl-2, COX-2, PI3K and AKT. This is the first report of anticancer mechanism involving 2,3-dihydroxy-9,10-anthraquinone in COLO320 cells. The present results might provide helpful suggestions for the design of antitumor drugs toward colorectal cancer treatment.

In vitro anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit.

The present study was undertaken to investigate the anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit and to explore the molecular mechanisms of action in MCF-7 cells. Cytotoxic properties of hexane, ethyl acetate and methanol extracts were carried out against MCF-7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Ethyl acetate extract showed good cytototoxic activities compared to hexane and methanol extracts. Methyl caffeate was isolated from the ethyl acetate extract using column chromatography. Cytotoxic properties of methyl caffeate was investigated against MCF-7, A549, COLO320, HepG-2 and Vero cells. The compound showed potent cytotoxic properties against MCF-7 cells compared to A549, COLO320 and HepG-2 cells. Methyl caffeate significantly reduced cell proliferation and increased formation of fragmented DNA and apoptotic body in MCF-7 cells. Bcl-2, Bax, Bid, p53, caspase-3, PARP and cytochrome c release were detected by western blot analysis. The activities of caspases-3 and PARP gradually increased after the addition of isolated compound. Bcl-2 protein was down regulated; Bid and Bax were up regulated after the treatment with methyl caffeate. Molecular docking studies showed that the compound bound stably to the active sites of poly (ADP-ribose) polymerase-1 (PARP1), B cell CLL/lymphoma-2 (BCL-2), E3 ubiquitin-protein ligase (MDM2) and tubulin. The results strongly suggested that methyl caffeate induced apoptosis in MCF-7 cells via caspase activation through cytochrome c release from mitochondria.

Patient-reported quality of life after allogeneic hematopoietic cell transplantation or chemotherapy for acute leukemia.

When discussing treatment options for patients with acute leukemia, it is important to acknowledge the impact of allogeneic hematopoietic cell transplantation (allo-HCT) or chemotherapy on quality of life (QOL). We performed a cross-sectional questionnaire study that administered SF-36, FACT-Leukemia and EuroQOL5D to 524 acute leukemia survivors, to compare patient-reported QOL between chemotherapy and allo-HCT, and to elucidate predictors of QOL. Patients who received chemotherapy alone had a better physical QOL than those who received allo-HCT. On the other hand, the allo-HCT group reported a better mental QOL. In the comparison of QOL in the allo-HCT patients according to the presence of GVHD at survey, patients who had GVHD symptoms experienced statistically and clinically significantly worse QOL than those who did not. In the allo-HCT patients without GVHD, the physical QOL was comparable to that in the chemotherapy patients, and they experienced significantly better mental and general QOL than the chemotherapy patients. GVHD and immunosuppressive drugs at survey were strongly associated with worse QOL after allo-HCT. In the chemotherapy group, a shorter time between treatment completion and survey was significantly associated with worse QOL. Further evaluation of QOL by a longitudinal assessment with quantitative and qualitative analyses are warranted.

Evaluation of the potassium adsorption capacity of a potassium adsorption filter during rapid blood transfusion.

The concentration of extracellular potassium in red blood cell concentrates (RCCs) increases during storage, leading to risk of hyperkalemia. A potassium adsorption filter (PAF) can eliminate the potassium at normal blood transfusion. This study aimed to investigate the potassium adsorption capacity of a PAF during rapid blood transfusion. We tested several different potassium concentrations under a rapid transfusion condition using a pressure bag. The adsorption rates of the 70-mEq/l model were 76·8%. The PAF showed good potassium adsorption capacity, suggesting that this filter may provide a convenient method to prevent hyperkalemia during rapid blood transfusion.

Delayed hematopoietic recovery after auto-SCT in patients receiving arsenic trioxide-based therapy for acute promyelocytic leukemia: a multi-center analysis.

A potential link between arsenic (ATO)-based therapy and delayed hematopoietic recovery after autologous hematopoietic SCT (HSCT) for acute promyelocytic leukemia (APL) has previously been reported. We retrospectively reviewed the clinical histories of 58 patients undergoing autologous HSCT for APL at 21 institutions in the United States and Japan. Thirty-three (56%) of the patients received ATO-based therapy prior to stem cell collection. Delayed neutrophil engraftment occurred in 10 patients (17%): 9 of the 10 patients (90%) received prior ATO (representing 27% of all ATO-treated patients), compared with 1 of the 10 patients (10%) not previously treated with ATO (representing 4% of all ATO-naïve patients; P<0.001). Compared with ATO-naïve patients, ATO-treated patients experienced significantly longer times to ANC recovery (median 12 days vs 9 days, P<0.001). In multivariate analysis, the only significant independent predictor of delayed neutrophil engraftment was prior treatment with ATO (hazard ratio 4.87; P<0.001). Of the available stem cell aliquots from APL patients, the median viable post-thaw CD34+ cell recovery was significantly lower than that of cryopreserved autologous stem cell products from patients with non-APL AML. Our findings suggest that ATO exposure prior to CD34+ cell harvest has deleterious effects on hematopoietic recovery after autologous HSCT.

Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H.

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and ß2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.