Pre-transplant donor-type red cell transfusion is a safe and effective strategy to reduce isohemagglutinin titers and prevent donor marrow infusion reactions in major ABO-mismatched transplants.

ABO incompatibility is not a barrier to allogeneic stem cell transplant but may result in acute hemolytic reactions. As stem cell product manipulation is cumbersome, we are reporting the effectiveness and safety of donor-type red cell infusion as a method of reducing acute hemolytic reaction while using marrow as stem cell source. In major ABO-mismatched bone marrow transplants, manipulation of marrow product requires expertise and expensive equipment, which may not be readily available to transplant centers in low- and middle-income regions. The aim behind our study is to report a safe and effective strategy to reduce isohemagglutinin titers and prevent donor marrow infusion reactions in major ABO-mismatched transplants. We retrospectively analyzed 303 consecutive allogeneic bone marrow transplants (BMTs) for beta thalassemia major, between August 2015 and March 2020, with either major (n = 41) or bidirectional (n = 14) mismatches. When isohemagglutinin titers were 1:32 or higher, donor-type packed red blood cell was divided into 4 aliquots, irradiated and administered over 4 days at incremental volumes. Patients were observed for hemolytic reaction, and if no reaction, bone marrow was infused without manipulation. Out of 55 patients, 20 received donor-type blood infusion. Twelve patients showed evidence of mild hemolysis. None developed severe hemolytic or anaphylactic reaction. Titers were rechecked in 14 patients and all had reduction in titers, except for one. Our experience demonstrated that donor-type PRBC infusion is safe and effective in preventing acute hemolysis in major ABO-mismatched stem cell transplants even with bone marrow as graft source.

Splenomegaly May Increase the Risk of Rejection in Low-Risk Matched Related Donor Transplant for Thalassemia, This Risk Can Be Partially Overcome by Additional Immunosuppression during Conditioning.

Severe thalassemia syndromes (ST) are highly curable by bone marrow transplant (BMT), but rejection may still occur. We retrospectively analyzed our fully matched related donor transplants to establish if isolated splenomegaly is an independent risk factor for rejection and if this risk can be reduced by modifying the conditioning protocol. In this study, we compared rejection rates between patients with and without splenomegaly in 189 consecutive low-risk ST transplants across 2 sequential conditioning regimens: regimen A (August 2013 to December 2016): busulfan (14 mg/kg oral, not adjusted to serum levels), cyclophosphamide (200 mg/kg), and anti-thymocyte globulin (ATG) (Genzyme (Sanofi, Paris, France) 4 mg/kg or Fresenius (Grafalon, Neovii Biotech GmbH, Gräfelfing Germany) 16 mg/kg on days -12 to -10), and regimen B: same backbone as regimen A except fludarabine total dose of 150 mg was added upfront and ATG dose was increased to 7 mg/kg in case of splenomegaly and/or sex-mismatched transplants (January 2017 to September 2018). Compared with regimen A, in regimen B, both overall rejection rates (RRs) (16% versus 6.5%, P = .023) and treatment-related mortality (TRM) (9.9% versus 2.8%, P = .038) improved significantly. By Cox regression analysis, the improvement in RR between the 2 protocols was particularly significant in patients with splenomegaly (RR 54.5% versus 6.5%, P = .00015; TRM 18.2% versus 6.5%, P = .25) (hazard ratio, 4.13; confidence interval, 1.61 to 10.6; P = .003). The increased risk of rejection related to splenomegaly can be overcome by adding fludarabine to the standard ATG-Busulfan- Cyclophosphamide (ATG-Bu-Cy) protocol without significantly increasing transplant-related morbidity and mortality or resorting to splenectomy pre-BMT.

Safety and Efficacy of Thalidomide and Hydroxyurea Combination in Beta Thalassemia Patients.

Beta thalassemia results from imbalance in alpha and beta globin chains causing severe anemia, transfusion dependency, and iron overload. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment. Patients without the option of HSCT may benefit from Hemoglobin F (HbF) inducing agents like thalidomide and hydroxyurea (HU). We conducted a retrospective analysis on 87 beta thalassemia patients who received a combination of low dose thalidomide and HU from January 2017 to December 2020. Patients received combination of HU 500 mg everyday (> 30 kg) or every alternate day (< 30 kg) and thalidomide 100 mg (> 30 kg) or 50 mg (< 30 kg) once daily. Parameters such as transfusion requirement, anthropometry, Hb levels, ferritin, drug side effects etc. were monitored and evaluated at the end of one year of therapy. Sixty-three patients (72%) achieved transfusion independence and were eligible for the study. Median time to transfusion independence was 6 months (range 3-11 months). At the end of 1 year, overall response rate was 72%. There was significant improvement in the Hb levels, ferritin values and height at the end of 1 year of follow up. No grade 3 or 4 toxicities were noted. We document improvement of Hb levels, transfusion independence, reduction in iron overload, and improvement in growth parameters with minimal side effects at the end of 1 year of follow up.

Bone Marrow Quality Index: A Predictor of Acute Graft-versus-Host Disease in Hematopoietic Stem Cell Transplantation for Thalassemia.

Bone marrow (BM) continues to be the preferred source of stem cells in allogenic transplantation for nonmalignant disorders. Granulocyte colony-stimulating factor (G-CSF)-primed BM is associated with low rates of acute graft-versus-host disease (aGVHD) and allows reduced collection volumes while ensuring speedy engraftment. However, variability in BM harvest quality is a concern. This study evaluated the utility of a novel indicator, the Bone Marrow Quality Index (BMQI), to predict aGVHD. We analyzed 184 consecutive first matched related donor bone marrow transplants for thalassemia using G-CSF-primed bone marrow over 6 years from March 2017 to April 2023 across 2 centers in India. BMQI was defined as the ratio of the G-CSF-primed BM WBC count to the peripheral blood WBC count within 24 hours of harvest. European Society for Blood and Marrow Transplantation criteria were used to grade aGVHD. The log-rank test was used to assess the impact of BMQI on aGVHD. The chi-square test was used to compare categorical data, and the Wilcoxon rank-sum test was used to compare the numerical data. A Cox proportional hazards model was used to investigate the association of BMQI vis-à-vis other factors on aGVHD. Of the 184 patients studied, 19 had a BMQI <.9, 18 had a BMQI between .9 and 1, and the remaining 147 had a BMQI >1. The rate of aGVHD grade II-IV was 37% in patients with a BMQI <.9 , 22% in those with BMQI .9 to 1, and 12% in those with BMQI >1 (P = .018). Patients with BMQI <.9 had a 3.1-fold greater chance (95% confidence interval [CI], .9 to 10.6) and those with BMQI .9 to 1 had a 2-fold greater chance (95% CI, .5 to 6.6) of developing aGVHD grade II-IV. BMQI was the significant predictor associated with aGVHD hazard (P = .014). BMQI appears to be the most relevant and controllable predictor of aGVHD. It is a novel, informative, and very simple indicator that could influence aGVHD prophylaxis decision making. Our indicator is accurately measurable, inexpensive, precise, and timely; furthermore, it does not involve any sophisticated equipment and thus may be widely applicable. Prior knowledge of poor BM quality may help intensify prophylaxis and monitoring for aGVHD, as well as trigger a review of collection procedures.

Do Weekly Surveillance Cultures Contribute to Antibiotic Stewardship and Correlate with Outcome of HSCT in Children? A Multicenter Real-World Experience of 5 Years from the Indian Subcontinent.

The utility of weekly rectal swab surveillance cultures (RSSCs) as a resource to identify gut colonization with extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli or Klebsiella pneumoniae carbapenemase (KPC)-producing organisms and guide empirical antibiotic therapy in hematopoietic stem cell transplantation (HSCT) recipients continues to be a subject of interest. There is an urgent need to assess and justify modifications to empirical antibiotics based on regional epidemiology and patient groups. This study aimed to study the utility of weekly rectal swab surveillance cultures (RSSCs) to guide empirical antibiotic therapy and to examine the impact of gut colonization on transplantation outcomes. This retrospective analysis of 317 successive first HSCTs performed mainly for hemoglobinopathies was conducted in 3 pediatric bone marrow transplantation centers in the Indian subcontinent between April 2016 and April 2021. Transplantation, infection control, and febrile neutropenia management protocols were identical in the 3 centers. First-line antibiotics were chosen based on RCCS reports, with meropenem used for ESBL and high-dose meropenem with colistin used for carbapenemase-resistant colonization for first half of the study, with no adjustment made in the second half. Clinical response to antibiotics, long-term outcomes, antibiotic-resistant bacteremia, and acute graft-versus-host disease (GVHD) were analyzed. The log-rank test, chi-square, and Wilcoxon rank-sum tests were used to compare data using R Statistical software. Of the 871 weekly RSSCs done, 162 were positive for ESBL- or KPC-resistant organism. RCCSs were ESBL-positive in 106 patients (33%) and KPC-positive in 10 patients (3%). Among the 97 ESBL-positive patients for whom a antimicrobial susceptibility testing report was available, only 22 (25%) demonstrated clinical resistance to piperacillin-tazobactam (Pip-Taz). Among the 10 KPC-positive patients, only 4 (40%) demonstrated clinical resistance to Pip-Taz and 3 (30%) had clinical resistance to meropenem. Two-thirds of patients with ESBL-positive RSSC in whom first-line empirical antibiotics were used responded clinically. Even among the 15 patients who were resistant to first-line empirical antibiotics (Pip-Taz) on RSSC reports, 67% responded clinically to Pip-Taz. Twenty-seven of these patients (56%) never needed carbapenem therapy. Empirical Pip-Taz therapy in ESBL-positive patients did not prolong meropenem use within 100 days of transplantation (P = .18). All patients with a KPC-positive RSSC who received first-line empirical antibiotics responded clinically, including 4 who were resistant to Pip-Taz and 3 who were meropenem-resistant on RCCS. Comparing patients who were ESBL-positive, KPC-positive, and not positive for either showed no statistically significant differences in overall survival (OS) (P = .95), disease-free survival (DFS) (P = .45), transplantation-related mortality (TRM) (P = .97), graft rejection (P = .68), or rate of acute GVHD grade II-IV (P = .78). No statistically significant differences were seen between the ESBL-positive patients who received and those who did not receive higher-level empirical antibiotics in OS (P = .32), DFS (P = .64), TRM (P = .65), graft rejection (P = .46), acute GVHD grade II-IV (P = .26), or antibiotic-resistant bacteremia (P = .3). In the context of HSCT for nonmalignant hematologic disorders, choosing empiric antibiotic therapy based on RSSCs is not justified, even in regions with a high prevalence of antimicrobial resistance. Antimicrobial susceptibility testing reports in surveillance cultures did not correlate with in vivo clinical response. Colonization reported on weekly RSSCs showed no correlation with clinical outcomes. © 2021 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Is it safe and efficacious to remove central lines in pediatric bone marrow transplant patients with platelets less than 20,000/µl?

Background: Patients with tunneled central venous lines (CVL) may develop bloodstream infections which at times are difficult to control without line removal. Concomitant severe thrombocytopenia with platelet transfusion refractoriness is often considered a major contraindication to any procedure involving a major blood vessel. There is very little literature on the clinical risks of tunneled central line removal in febrile pancytopenia patients. Procedure: We analyzed complications and outcomes in all our patients, a total of 52, who underwent CVL removal with platelets <20,000/µl. Results: CVL removal was done on a median day of 17.5 with 47 of the 52 patients never having achieved platelets engraftment prior to line removal. No bleeding episodes or unplanned transfusions could be associated with CVL removal. No other complications were also reported. All patients had time to hemostasis within 5 min of catheter removal. Removal of CVL under local anesthesia remained complication-free even at platelet counts less than 20,000/ul. A total of 31 patients were febrile at the time of CVL removal, of which 17 became afebrile within 2 days. We found no difference in defervescence when comparing those whose antibiotic therapy was changed/escalated versus those in whom it was not. Conclusion: Our findings suggest that central lines can be safely removed with platelet counts less than 20,000/ul and that this may result in enhanced bloodstream infection control. This might be particularly relevant to neutropenic patients in this day and age of multidrug-resistant organism emergence and paucity of new effective antibiotics.

A panel of artificial APCs expressing prevalent HLA alleles permits generation of cytotoxic T cells specific for both dominant and subdominant viral epitopes for adoptive therapy.

Adoptive transfer of virus-specific T cells can treat infections complicating allogeneic hematopoietic cell transplants. However, autologous APCs are often limited in supply. In this study, we describe a panel of artificial APCs (AAPCs) consisting of murine 3T3 cells transduced to express human B7.1, ICAM-1, and LFA-3 that each stably express one of a series of six common HLA class I alleles. In comparative analyses, T cells sensitized with AAPCs expressing a shared HLA allele or autologous APCs loaded with a pool of 15-mer spanning the sequence of CMVpp65 produced similar yields of HLA-restricted CMVpp65-specific T cells; significantly higher yields could be achieved by sensitization with AAPCs transduced to express the CMVpp65 protein. T cells generated were CD8(+), IFN-gamma(+), and exhibited HLA-restricted CMVpp65-specific cytotoxicity. T cells sensitized with either peptide-loaded or transduced AAPCs recognized epitopes presented by each HLA allele known to be immunogenic in humans. Sensitization with AAPCs also permitted expansion of IFN-gamma(+) cytotoxic effector cells against subdominant epitopes that were either absent or in low frequencies in T cells sensitized with autologous APCs. This replenishable panel of AAPCs can be used for immediate sensitization and expansion of virus-specific T cells of desired HLA restriction for adoptive immunotherapy. It may be of particular value for recipients of transplants from HLA-disparate donors.

Comparison of Adverse Outcomes Following Placement of Superion Interspinous Spacer Device Versus Laminectomy and Laminotomy.

BACKGROUND: Current evidence suggests placement of the Superion interspinous spacer (SISS) device compared with laminectomy or laminotomy surgery offers an effective, less invasive treatment option for patients with symptomatic lumbar spinal stenosis. Both SISS placement and laminectomy or laminotomy have risks of complications and a direct comparison of complications between the 2 procedures has not been previously studied. The purpose of this study is to compare the short-term complications of the SISS with laminectomy or laminotomy and highlight device-specific long-term outcomes with SISS. METHODS: Via retrospective review, 189 patients who received lumbar level SISSs were compared with 378 matched controls who underwent primary lumbar spine laminectomy or laminotomy; data were collected from the American College of Surgeons National Surgical Quality Improvement Program database. Complications analyzed included rates of wound infection, pulmonary embolism, deep venous thrombosis, urinary tract infection, sepsis, septic shock, cardiac arrest, death, and reoperation within 30 days of index surgery. Differences between groups were analyzed using the χ2test. Device-specific complication (DSC) rates included device malfunction or misplacement (DM), device explantation (DE), spinous process fracture (SPF), and subsequent spinal surgery (SSS). RESULTS: No differences in demographics or comorbidities existed between groups. There was no significant difference in rates of complications between groups. A total of 44.4% of patients in the SISS group experienced DSCs with 11.1% of patients experiencing DM, 21.1% experiencing an SPF, 20.1% requiring DE, and 24.3% requiring SSS. Having at least 1 DSC significantly increased odds of SSS, odds ratio >120, P < .0001. CONCLUSION: Rates of 30-day complications in the SISS group were not significantly different from patients undergoing laminectomy or laminotomy. Rates of 2-year DSC within SISS and cumulative risk associated with these complications should be considered further as they likely represent need for additional procedures for patients and substantial cost to the healthcare system. LEVEL OF EVIDENCE: 4. CLINICAL RELEVANCE: Having no differences in adverse events between laminectomies or laminotomies and SISS plus evidence of substantial device-specific long-term adverse outcomes and reoperation should be given consideration when deciding on surgical intervention of 1-2 level lumbar spinal stenosis.

Setting up and sustaining blood and marrow transplant services for children in middle-income economies: an experience-driven position paper on behalf of the EBMT PDWP.

Severe blood disorders and cancer are the leading cause of death and disability from noncommunicable diseases in the global pediatric population and a major financial burden. The most frequent of these conditions, namely sickle cell disease and severe thalassemia, are highly curable by blood or bone marrow transplantation (BMT) which can restore a normal health-related quality of life and be cost-effective. This position paper summarizes critical issues in extending global access to BMT based on ground experience in the start-up of several BMT units in middle-income countries (MICs) across South-East Asia and the Middle East where close to 700 allogeneic BMTs have been performed over a 10-year period. Basic requirements in terms of support systems, equipment, and consumables are summarized keeping in mind WHO's model essential lists and recommendations. BMT unit setup and maintenance costs are summarized as well as those per transplant. Low-risk BMT is feasible and safe in MICs with outcomes comparable to high-income countries but at a fraction of the cost. This report might be of assistance to health care institutions in MICs interested in developing hematopoietic stem cell transplantation services and strengthening context appropriate tertiary care and higher medical education.

Life expectancy and risk factors for early death in patients with severe thalassemia syndromes in South India.

In spite of advances in chelation therapy and screening of blood, mortality associated with the most common life-threatening noncommunicable disease of children in India, transfusion-dependent thalassemia (TDT), remains poorly defined. This study aims at estimating death rates and mortality risk factors associated with TDT. The clinical records of 1087 patients from 5 thalassemia centers in India were retrospectively analyzed from 2011 to 2018. Median patient age was 8.5 years, with 107 patients older than 18 years; 656 patients were male and 431 were female. Demographic details and clinical parameters were analyzed at presentation and at last visit. With 41 recorded deaths, actuarial survival at 26.9 years was 50%, and under-5 mortality was 7 times higher than in the general population. Patients with transfusion-transmitted infections (TTIs) had 3.4 times higher risk for death (P = .031). Serum ferritin higher than 4000 ng/dL had 4.6 times higher risk for mortality compared with ferritin lower than 1000 ng/dL (P = .00063). A hemoglobin drop lower than 2 g/dL per week had 7.7 times higher mortality risk compared with a drop of less than 1 g/dL per week (P < .0001). Social determinants (sex, economic status, and distance from center), splenectomy, and even cardiac complications were not associated with higher mortality risk. Main causes of death were infection, iron overload, TTIs, and allo-immunization. Patients who received more than 4 years of adequate care had more than 66% mortality risk reduction (P < .0001). TDT in India continues to result in high mortality. Ineffective transfusion, TTIs, and chelation continue to be the most significant risk factors. Comprehensive care in dedicated day care centers from early age is likely to improve outcomes.

Adoptive transfer of antigen-specific T-cells of donor type for immunotherapy of viral infections following allogeneic hematopoietic cell transplants.

Allogeneic marrow and cytokine-mobilized peripheral blood stem cells adequately depleted of T cells prevent acute and chronic forms of graft versus host disease in HLA-matched and non-identical hosts without any posttransplant immunosuppressive prophylaxis. Current cytoreductive regimens secure consistent durable engraftment, and full donor chimerism. The risk of relapse following such transplants in patients with AML and ALL has been low, and not different from that recorded following unmodified transplants. However, in HLA-disparate hosts the risk of infections caused by EBV, CMV, and certain fungi are increased. To address this limitation, others and we are exploring adoptive immunotherapies with in vitro generated, pathogen-specific T cells. Early clinical trials already indicate the potential of such T cells to treat and prevent life threatening diseases caused by these pathogens, particularly in recipients of T cell depleted grafts who do not require ongoing treatment with immunosuppressive agents, and therefore provide a permissive environment for the expansion and persistence of the T cells following adoptive transfer. New more predictable strategies are under development, which should allow such therapies to be broadly applicable.

Ocimum sanctum modulates selenite-induced cataractogenic changes and prevents rat lens opacification.

PURPOSE: To study the effect of Ocimum sanctum (OS) on selenite-induced morphological and biochemical changes in isolated rat lenses as well as on cataract incidence in rat pups. METHODS: Transparent rat lenses were divided into normal, selenite-only, and four treated groups. Selenite-only and treated group lenses were subjected to oxidative stress in vitro by incorporating sodium selenite (100 microM) in the culture medium. The effect of OS (70, 140, 280, and 560 microg/ml) was studied on the levels of reduced glutathione (GSH) and thiobarbituric acid reacting substances (TBARS) in selenite-challenged lenses. The lowest concentration of OS offering significant modulation on these two parameters was determined. Subsequently, the effect of prior and cotreatment with the lowest effective concentration of OS was studied on TBARS, GSH, and on lens antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), catalase (CAT), and glutathione-S-transferase (GST). Changes in lens protein profiles under different incubation conditions were analyzed by SDS gel-electrophoresis. In vivo, cataract was induced by a single subcutaneous injection of sodium selenite (25 micromole/kg b.w.) to 9-day-old rat pups. The anticataract effect of OS (5 and 10 mg/kg b.w.) injected intraperitoneally 4 hr prior to selenite challenge was evaluated by the presence of lens nuclear opacity in rat pups on the 16th postnatal day. Insolubilization of lens proteins post-selenite injection was monitored for 4 days. RESULTS: The lenses in the selenite-only group developed cortical opacities in 24 hr. OS showed different degrees of positive modulation in selenite-induced morphological as well as biochemical changes. The lowest effective dose of OS that significantly modulated glutathione and thiobarbituric acid reacting substances was found to be 140 microg/ml. At this dose, a significant increase in antioxidant enzyme levels and preservation of normal lens protein profile was observed. OS at the dose of 70 microg/ml did not show any significant protection with respect to either morphology or biochemistry of lenses. In vivo, 5 and 10 mg/kg of OS reduced the incidence of selenite cataract by 20% and 60%, respectively, and prevented protein insolubilization as well. CONCLUSIONS: Aqueous extract of OS possesses potential anticataract activity against selenite-induced experimental cataractogenesis. The protective effect was supported by restoration of the antioxidant defense system and inhibition of protein insolubilization of rat lenses as well.

Lycopene attenuates oxidative stress induced experimental cataract development: an in vitro and in vivo study.

OBJECTIVES: Lycopene, a nutritional antioxidant, was evaluated for its anticataract potential to further establish its role in cataract prevention. METHODS: The ability of lycopene to modulate the biochemical parameters was investigated by in vitro studies. Enucleated rat lenses were maintained in organ culture containing Dulbecco's Modified Eagles Medium alone or in addition with 100 microM selenite and served as the normal and control groups, respectively. For the test group, the control medium was supplemented with 10 microM lycopene. The lenses were incubated for 24 h at 37 degrees C. At the end of the incubation period, the lenses were examined for morphologic variation, and biochemical parameters such as reduced glutathione, the lipid peroxidation product malondialdehyde, and the antioxidant enzymes glutathione peroxidase, glutathione S-transferase, superoxide dismutase, and catalase were estimated. In vivo selenite cataract was induced in 9-d-old rats by subcutaneous injection of sodium selenite (25 micromoles/kg of body weight). The rats in the test group were injected with lycopene (200 microg/kg body weight, intraperitoneally) 4 h before the selenite challenge. The incidence of cataract was observed when the rats first opened their eyes. Galactose cataract was induced in rats by feeding 30% galactose in the diet. Rats in the test group were fed orally with 200 microg/kg of lycopene daily, and rats in the control group received only vehicle. Cataract stages were graded at regular intervals. RESULTS: A fall (25%) in the glutathione level and a rise (32%) in the malondialdehyde content were observed in control as opposed to normal lenses. Lycopene supplementation in the medium significantly (P < 0.001) restored glutathione and malondialdehyde levels. A significant decrease in the activity of antioxidant enzymes also was observed in the control lenses. A significant restoration in the activities of superoxide dismutase (P < 0.05) and catalase and glutathione S-transferase (P < 0.01), with no effect on glutathione peroxidase, was observed in the lycopene-supplemented group. Lycopene also reduced the incidence of selenite cataract. Only 9% of the eyes in the test group developed dense nuclear opacity as opposed to 83% in the control group. A significant delay in the onset and progression of galactose cataract was observed with oral feeding of lycopene. Only 35% of the eyes developed mature cataract as opposed to 100% in the control group. CONCLUSIONS: Lycopene protects against experimental cataract development by virtue of its antioxidant properties, and it may be useful for prophylaxis or therapy against cataracts.

Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy.

Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We used a pool of 138 synthetic overlapping pentadecapeptides overspanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T cells that selectively recognized 1-3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide-loaded targets sharing single HLA class I or II alleles identified the restricting HLA alleles. HLA-A*0201+ donors consistently responded to pentadecapeptides containing HLA-A*0201-binding epitope(aa495-503)NLVPMVATV. T-cell lines from other donors contained high frequencies of CD4 and/or CD8 T cells selectively reactive against peptides presented by other HLA alleles, including both known epitopes such as (aa341-350)QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as (aa267-275)HERNGFTVL (HLA-B*4001 and B*4002) and (aa513-523)FFWDANDIYRI (HLA-DRB1*1301). These T cells consistently lysed CMV-infected target cells. Thus, this approach fosters expansion and selection of HLA-restricted CMV-pp65-reactive T-cell lines of high specificity that also lyse CMV-infected targets, and from a functional and regulatory perspective, may have advantages for generating virus-specific T cells for adoptive immunotherapy.

21. Immunohematologic disorders.

Immunohematologic disorders are a broad group of entities in which hematologic diseases, usually cytopenias, are caused by immune reactions. These reactions lead to the development of hemolytic anemia, thrombocytopenia, or neutropenia, either separately or in combination. Common underlying mechanisms include immunodeficiency, systemic autoimmunity, and drug-induced reactions. Transplacental transfer of maternal alloantibodies can lead to fetal and neonatal cytopenias. Other immune reactions include antibodies to clotting factors, which result in both thrombosis and hemorrhage, and hemolytic and nonhemolytic reactions during transfusion of blood products.

Impact of environmental pollution on the eye.

PURPOSE: To study the effect of environmental toxins on the ocular surface in persons travelling through highly polluted areas of the metropolis of Delhi. METHODS: A total of 500 apparently healthy volunteers recruited from the metropolis of Delhi were examined to establish the frequency of ocular surface disorders. All subjects underwent a slit-lamp examination, tear break-up time (BUT) test, Rose Bengal test, Schirmer's test and tear lysozyme level test. RESULTS: Subjects in the study group had significantly higher levels of ophthalmic symptoms compared to the control group. In all, 105 (42%) and 125 (50%) subjects in the study group compared to 50 (20%) and 65 (26%) in the control group complained of redness and irritation (p < 0.05). There was no significant difference in visual acuity (VA) between the two groups, with best corrected VA being 6/9 or better in 92% of the study group and 96% of the control group. The average (+/-standard deviation) Schirmer's test result was 13.42+/-6.67 mm in the study group compared to 15.95+/-6.14 mm in the control group. This difference was statistically significant. The BUT was also significantly lower in the study group (12.97+/-6.12 seconds) compared to the control group (19.23+/-5.70 seconds) (p < 0.001). The Rose Bengal test and tear lysozyme activity did not show any significant difference between the two groups. CONCLUSION: According to our study, environmental conditions appear to have a very significant effect on the ocular surface. There were very high levels of subclinical ocular surface changes among persons travelling in highly polluted areas.

Lycopene prevents sugar-induced morphological changes and modulates antioxidant status of human lens epithelial cells.

Cataract is a multifactorial disease. Osmotic stress, together with weakened antioxidant defence mechanisms, is attributed to the changes observed in human diabetic cataract. Epidemiological studies provide evidence that nutritional antioxidants slow down the progression of cataract. The usefulness of lycopene, a dietary carotenoid, in the pathogenesis of human cataracts has not been studied so far. Since the epithelium is the metabolic unit of the lens, the effect of lycopene on galactose-induced morphological changes and antioxidant status of human lens epithelial cells (HLEC) in culture was evaluated in the present study. HLEC of fresh cadaver eyes obtained from an eye bank were cultured in medium supplemented with fetal calf serum (200 ml/l). On confluency, the cells were subcultured in medium containing either 30 mm-d-galactose or 30 mm-d-galactose+lycopene (5, 10 or 20 microm) for 72 h. The cells were observed under the phase-contrast microscope and transmssion electron microscope for any morphological changes and then harvested for the estimation of various biochemical variables. Malondialdeyde, glutathione and antioxidant enzymes were significantly altered in the control as compared with the normal cultures. Vacuolization was also observed in the presence of galactose. Addition of lycopene confers significant protection against these changes in HLEC.

Pyruvate modulates antioxidant status of cultured human lens epithelial cells under hypergalactosemic conditions.

Lens epithelial cells are the metabolic unit of the lens and antioxidant enzymes are mainly concentrated here. The purpose of this study was to maintain human lens epithelial cells (HLEC) in culture and examine the status of antioxidant enzymes (glutathione peroxidase (GSHPx), catalase (CAT), glutathione-S-transferase (GST)), lipid peroxidation product malondialdehyde (MDA) and glutathione (GSH) levels in these cells under normal as well as hypergalactosemic (30 mM galactose) conditions. Further, effect of pyruvate, a physiological antioxidant has also been evaluated on these parameters. For conducting experiments, anterior capsule specimens obtained from fresh cadaver eyes from eye bank were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal calf serum. Upon confluency, the cells were subcultured in three separate flasks containing DMEM alone (normal group), DMEM + 30 mM D-galactose (control group), DMEM + 30 mM D-galactose + 5 mM pyruvate (test group) and incubated for 24 or 72 h. These cells were observed under the phase contrast microscope for any morphological changes and harvested for the estimation of various antioxidant parameters. Our results show significant weakened antioxidant defense in HLEC when incubated in the presence of galactose as compared to normal. Addition of pyruvate significantly modulated levels of GSH, MDA, GSHPx, CAT and GST.

Pyruvate inhibits galactosemic changes in cultured cat lens epithelial cells.

An attempt was made to maintain cat lens epithelial cells (CLEC) in culture and study the morphology, growth and survival of these cells in vitro. The influence of incorporation of galactose (30 mM) into the culture medium on the morphology and biochemistry of CLEC in the primary culture was then investigated. To establish the effect of galactose on CLEC, various biochemical parameters associated with galactosemic cataract such as aldose reductase (AR), Na+K+ATPase, glutathione, polyol and soluble/insoluble proteins were estimated after 24 h of incubation. The effect of pyruvate (5 mM), a 'physiological antioxidant', on the changes induced by galactose in CLEC was studied. CLEC in culture showed regular hexagonal cells with prominent nuclei. The CLEC culture attained confluency in 11 days during primary culture and semiconfluency in 14 days in two subsequent passages. Vacuolization and significantly raised AR activity, polyol levels and insoluble protein contents were observed; they had no effect on Na+K+ATPase and soluble protein after 24 h of incubation in the culture medium with galactose. Supplementation of pyruvate (5 mM) resulted in a lesser number of vacuoles together with a positive modulation of these parameters.