Activity of broad-spectrum T cells as treatment for AdV, EBV, CMV, BKV, and HHV6 infections after HSCT.

It remains difficult to treat the multiplicity of distinct viral infections that afflict immunocompromised patients. Adoptive transfer of virus-specific T cells (VSTs) can be safe and effective, but such cells have been complex to prepare and limited in antiviral range. We now demonstrate the feasibility and clinical utility of rapidly generated single-culture VSTs that recognize 12 immunogenic antigens from five viruses (Epstein-Barr virus, adenovirus, cytomegalovirus, BK virus, and human herpesvirus 6) that frequently cause disease in immunocompromised patients. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term.

Safety and clinical efficacy of rapidly-generated trivirus-directed T cells as treatment for adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant.

Adoptive transfer of virus-specific T cells can prevent and treat serious infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv) after allogeneic hematopoietic stem cell transplant. It has, however, proved difficult to make this approach widely available since infectious virus and viral vectors are required for T cell activation, followed by an intensive and prolonged culture period extending over several months. We now show that T cells targeting a range of viral antigens derived from EBV, CMV, and Adv can be reproducibly generated in a single culture over a 2-3-week period, using methods that exclude all viral components and employ a much-simplified culture technology. When administered to recipients of haploidentical (n = 5), matched unrelated (n = 3), mismatched unrelated (n = 1) or matched related (n = 1) transplants with active CMV (n = 3), Adv (n = 1), EBV (n = 2), EBV+Adv (n = 2) or CMV+Adv (n = 2) infections, the cells produced complete virological responses in 80%, including all patients with dual infections. In each case, a decrease in viral load correlated with an increase in the frequency of T cells directed against the infecting virus(es); both immediate and delayed toxicities were absent. This approach should increase both the feasibility and applicability of T cell therapy. The trial was registered at www.clinicaltrials.gov as NCT01070797.

Immunotherapeutic strategies to prevent and treat human herpesvirus 6 reactivation after allogeneic stem cell transplantation.

Human herpesvirus (HHV) 6 causes substantial morbidity and mortality in the immunocompromised host and has no approved therapy. Adoptive transfer of virus specific T cells has proven safe and apparently effective as prophylaxis and treatment of other virus infections in immunocompromised patients; however, extension to subjects with HHV6 has been hindered by the paucity of information on targets of cellular immunity. We now characterize the cellular immune response from 20 donors against 5 major HHV6B antigens predicted to be immunogenic and define a hierarchy of immunodominance of antigens based on the frequency of responding donors and the magnitude of the T-cell response. We identified specific epitopes within these antigens and expanded the HHV6 reactive T cells using a GMP-compliant protocol. The expanded population comprised both CD4(+) and CD8(+) T cells that were able to produce multiple effector cytokines and kill both peptide-loaded and HHV6B wild-type virus-infected target cells. Thus, we conclude that adoptive T-cell immunotherapy for HHV6 is a practical objective and that the peptide and epitope tools we describe will allow such cells to be prepared, administered, and monitored in human subjects.

Rapidly generated multivirus-specific cytotoxic T lymphocytes for the prophylaxis and treatment of viral infections.

Severe and fatal viral infections remain common after hematopoietic stem cell transplantation. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenoviral antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional viruses is limited by competition between virus-derived antigens and time-consuming and laborious manufacturing procedures. We now describe a system that rapidly generates a single preparation of polyclonal (CD4(+) and CD8(+)) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, Adenovirus (Adv), BK, human herpes virus (HHV)-6, respiratory syncytial virus (RSV), and Influenza) that commonly cause post-transplant morbidity and mortality. CTLs can be rapidly produced (10 days) by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines interleukin-4 (IL4) and IL7. This approach reduces the impact of antigenic competition with a consequent increase in the antigenic repertoire and frequency of virus-specific T cells. Our approach can be readily introduced into clinical practice and should be a cost-effective alternative to common antiviral prophylactic agents for allogeneic hematopoietic stem cell transplant (HSCT) recipients.

Engineered T cells for pancreatic cancer treatment.

OBJECTIVE: Conventional chemotherapy and radiotherapy produce marginal survival benefits in pancreatic cancer, underscoring the need for novel therapies. The aim of this study is to develop an adoptive T cell transfer approach to target tumours expressing prostate stem cell antigen (PSCA), a tumour-associated antigen that is frequently expressed by pancreatic cancer cells. METHODS: Expression of PSCA on cell lines and primary tumour samples was confirmed by immunohistochemistry. Healthy donor- and patient-derived T cells were isolated, activated in vitro using CD3/CD28, and transduced with a retroviral vector encoding a chimeric antigen receptor (CAR) targeting PSCA. The ability of these cells to kill tumour cells was analysed by chromium-51 (Cr(51)) release. RESULTS: Prostate stem cell antigen was expressed on >70% of the primary tumour samples screened. Activated, CAR-modified T cells could be readily generated in clinically relevant numbers and were specifically able to kill PSCA-expressing pancreatic cancer cell lines with no non-specific killing of PSCA-negative target cells, thus indicating the potential efficacy and safety of this approach. CONCLUSIONS: Prostate stem cell antigen is frequently expressed on pancreatic cancer cells and can be targeted for immune-mediated destruction using CAR-modified, adoptively transferred T cells. The safety and efficacy of this approach indicate that it deserves further study and may represent a promising novel treatment for patients with pancreatic cancer.

Amiloride improves locomotor recovery after spinal cord injury.

Amiloride is a drug approved by the United States Food and Drug Administration, which has shown neuroprotective effects in different neuropathological conditions, including brain injury or brain ischemia, but has not been tested in spinal cord injury (SCI). We tested amiloride's therapeutic potential in a clinically relevant rat model of contusion SCI inflicted at the thoracic segment T10. Rats receiving daily administration of amiloride from 24 h to 35 days after SCI exhibited a significant improvement in hindlimb locomotor ability at 21, 28, and 35 days after injury, when compared to vehicle-treated SCI rats. Rats receiving amiloride treatment also exhibited a significant increase in myelin oligodendrocyte glycoprotein (MOG) levels 35 days after SCI at the site of injury (T10) when compared to vehicle-treated controls, which indicated a partial reverse in the decrease of MOG observed with injury. Our data indicate that higher levels of MOG correlate with improved locomotor recovery after SCI, and that this may explain the beneficial effects of amiloride after SCI. Given that amiloride treatment after SCI caused a significant preservation of myelin levels, and improved locomotor recovery, it should be considered as a possible therapeutic intervention after SCI.

Improved resolution of diagnostic problems in selected prostate needle biopsy specimens by using the ASAP workup: a prospective study of interval sections vs new recut sections.

This study assessed the value of an atypical small acinar proliferation (ASAP) workup consisting of preparing new recut sections from the paraffin block and performing H&E-stained sections and immunostains (using the antibody cocktail for p63, cytokeratins 5 and 14, and alpha-methylacyl coenzyme A racemase) on the slides. We compared the ASAP workup with the interval workup, the common practice of performing the same immunostains on the saved interval sections, in 105 cases because of focal glandular atypia on the original H&E-stained sections. There were no specimens in which only the interval workup showed a changed diagnosis, but there were 23 specimens (21.9%) in which the preliminary diagnosis was changed to a definitive diagnosis of carcinoma (10 specimens) or a specific benign diagnosis (13 specimens) based solely on the findings of the ASAP workup. The ASAP workup is recommended as a very useful histologic tool for resolving diagnostic problems in prostate needle biopsy specimens.

CD5+ immunophenotype in the bone marrow but not in the peripheral blood in a patient with hairy cell leukemia.

We report the case of a patient with rare CD5+ hairy cell leukemia, which was unusual, as there was also a discrepancy between the bone marrow and peripheral blood immunophenotypes. We propose that some degree of maturation within the malignant clone and the predominance of more mature CD5- hairy cells in the peripheral blood can explain this observation. The patient was treated with a conventional course of cladribine (2'-chlorodeoxyadenosine) and achieved complete remission. We conclude that the unusual CD5+ immunophenotype of this patient did not affect prognosis or change the response to standard therapy.

The first year of clinical experience with concurrent post abortal insertion of copper "T" 200.

PIP: Immediate postabortion period is a strategic time for providing contraceptive protection, especially insertion of IUD concommitent with the abortion procedure. One can be reasonably safe in assuming that copper T 200 may be used with comparable degrees of clinical effectiveness in such situations. A higher incidence of IUD removals for bleeding and IUD displacements in the postabortal insertion is justifiable when it is considered that a greater number of women are reached and thus protected against the risks of an unwanted pregnancy. Major problems such as cervical or uterine perforations were not associated with this type of insertion. IUD removal was effected in 5.39% for bleeding, and in 4.69% of cases where there was either complete or incomplete expulsion of the device. Expulsion rate was high within the 1st week of insertion, and it is reasonable to believe that abortion complications were responsible for this high expulsion rate. Even though IUD removal was not required, about 15% of the patients had menstrual irregularities at the end of 1 year of use. Embedding of the copper device did not pose any problem for removal of the device after 1 year of use and no fragmentation of copper wire was found in the IUDs removed. In those who wanted, conception had occurred within 4 months of removal of the device. The follow-up data suggests that the use-effectiveness of the device gradually increase with the duration of use and is found to be highest after 1 year of use.^ieng