Effect of fetal liver condition media derived cytokines(IL-6 and Flt-3) on human bone marrow stem cells colony formation.

Earlier research from our laboratory demonstrated the presence of stimulatory activity of different growth factors in the fetal liver (FL) extracts when collected in a medium known as fetal liver conditioned medium (FLCM) using Enzyme-linked Immunosorbent Assay (ELISA). In the present study, we have assessed two other cytokines viz. IL-6 and FMS like tyrosine kinase-3 (Flt-3) with the help of bioneutralization assay. FLCM was prepared by incubating fetal liver cells with Iscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal bovine serum (FBS) and 10% Phytohemagglutinin and collected after 24hrs, 48hrs, 72 hrs. and on the 7th day of incubation. Clonal cultures were established for 1 X 105 normal bone marrow (BM) mononuclear cells (NBM MNC) per plate with methylcellulose medium containing cytokines SCF and EPO. Mean Colony forming units-granulocytes, erythrocytes, macrophages, megakaryocytes (CFU-GEMM) were assessed with and without the addition of FLCM. It was found that FLCM enhanced the number of colonies made by NBM MNCs. Further, cytokines IL-6 and Flt-3, present in FLCM, were bioneutralized with respective anti-cytokine antibodies. Neutralized FLCM was evaluated for the colony-forming potential of CFU-GEMM colonies. The maximum reduction of 42% was seen with 20 ng/ml of anti-IL-6 antibody. Maximum suppression up to 20% was observed with 0.7 ng/ml of anti Flt-3 antibody for CFU-GEMM colonies. Presence of cytokines IL-6 and Flt-3 in FL extracts and their colony stimulatory activity suggests that fetal liver infusion (FLI) may be a valuable alternative for managing BM recovery in certain clinical conditions such as AA.

In vitro expansion of fetal liver hematopoietic stem cells.

Fetal liver hematopoietic stem and progenitor cells (HSPCs) have been considered appropriate for the management of aplastic anemia owing to their proliferative potential. Bone marrow recovery was possible in some cases; the engraftment potential of these cells, however was unsatisfactory, possibly due to the availability of a smaller number of these cells from a single fetus. The present study explores how we can expand fetal liver hematopoietic stem cells under in vitro conditions. We isolated mononuclear cells from fetal liver and hematopoietic stem cells were identified and analyzed by cell surface marker CD34. CD34+ fetal liver HSPCs cells were separated by magnetic cell sorting positive selection method. HSPCs (CD34+) were cultured by using 5 cytokines, stem cell factor (SCF), granulocyte macrophages-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), Fms-related tyrosine kinase 3 (FLT-3) and erythropoietin (EPO), in 4 different combinations along with supplements, in serum-free culture media for 21 days. Cell viability continued to be greater than 90% throughout 21 days of culture. The cells expanded best in a combination of media, supplements and 5 cytokines, namely SCF, FLT-3, IL6, EPO and GM-CSF to yield a large number of total (CD34+ & CD34-) cells. Even though the total number of nucleated cells increased in culture significantly, levels of CD34 antigen expression declined steadily over this period.

The short-term impact of protocol biopsies in a live-related renal transplant program using tacrolimus based immunosuppression.

The aim of the study was to assess the impact of protocol biopsies in a live-related renal transplant program using tacrolimus-based immunosuppression in the short term. Eighty-three live-related transplant recipients were randomly allocated to protocol biopsy group (Group I, n = 40) and a control group (Group II, n = 43). Other immunosuppressants in these groups consisted of either mycophenolate mofetil or azathioprine and steroids. Protocol biopsies were conducted in biopsy group at 1, 6, and 12 months post-transplant. The non-biopsy group was followed by serial serum creatinine and biopsies in them were conducted as and when clinically indicated. Both groups were analyzed at 12 months with respect to graft function and survival. The two groups were similar with respect to age, number of dialysis pre-operatively, tacrolimus levels, induction therapy, donor age, and donor glomerular filtration rate. Forty protocol biopsies were conducted at 1 month, 31 at 6 months, and 26 at 12 months. The prevalence of sub-clinical rejection at 1, 6, and 12 months in these biopsies was 17.5%, 11.2%, and 10.3%, respectively. The prevalence of calcineurin inhibitor toxicity during same period was 15%, 15.5%, and 14.4%, respectively. The cumulative rejection rate in Group I and Group II at 12-month follow-up was 10.3% and 11.3% (P = 0.78), respectively, and cumulative calcineurin inhibitor toxicity at 12 months was 14.4% and 9.3% (P = 0.59), respectively, were not statistically significant. There was no difference in graft survival and function at 1 year. Protocol biopsies have a limited role in a well-matched renal transplant program with tacrolimus-based immunosuppression in the short term. However, the long-term impact of protocol biopsies needs further evaluation.

Role of killer immunoglobulin-like receptor-ligand interactions in human leukocyte antigen-matched sibling hematopoietic stem cell transplantation.

INTRODUCTION: Killer immunoglobulin-like receptor (KIR)-ligand mismatches lead to natural killer cell alloreactivity after hematopoietic stem cell transplantation (HSCT). However, their clinical impact on HSCT outcomes is controversial due to complexity of KIR haplotypes, genotypes, and phenotypes as well as their diversity among patient populations. The present study investigated the role of KIR-ligand interactions in human leukocyte antigen (HLA)-matched sibling transplants. METHODS: The recipient cohort, which included patients diagnosed with aplastic anemia, acute leukemia, and myelodysplastic syndrome, received granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood stem cells. HLA typing was performed using polymerase chain reaction - sequence specific oligo probes (PCR-SSO). The KIR genotype of the donors and the ligands C1 (Asparagine 80), C2 (Lysine 80), and Bw4 recipient typings were performed using polymerase chain reaction - sequence specific primers (PCR-SSP). We assessed acute and chronic graft-versus-host disease (GVHD), relapse, and overall survival. RESULTS: While 84.5% of donors carried a Bx KIR, 15.5% carried the AA haplotype. The effect of a recipient's lack of ligands among 88.5% of cases was associated with 39% of subjects developing GvHD. Lack of C1 may lead to manifestations of acute GvHD and lack of C2 to manifestation of chronic GvHD. The presence of both C1 and C2 seemed to be protective against both forms of GvHD. The role of two Bw4 alleles, threonine (T) or isoleucine (I) at position 80, was evaluated. 73% of recipients who carried Bw4 80(I) versus 27% with the Bw4 80(T) allele. The presence of Bw4-80(T) allele appeared to reduce the risk of GvHD, indicating its stronger inhibitory effect than its 80(I) counterpart. CONCLUSION: KIR-ligand interactions influenced HSCT outcomes.

Outcome of pediatric renal transplantation in north India.

We report our experience and long-term outcome of pediatric renal transplantation at a referral center in New Delhi. During 1995-2008, 45 transplants were performed in 43 patients at a mean age of 13.3 ± 4.0 (range 3.8-18) yr. The chief causes for ESRD were reflux nephropathy, obstructive uropathy, vasculitis, renal dysplasia, and focal segmental glomerulosclerosis. Most (91.1%) donors were living related. Post-transplant immunosuppression comprised prednisolone, a calcineurin inhibitor and azathioprine or MMF. AR and CR were seen in 14 (31.1%) and 12 (26.7%) allografts, respectively. Predictors of CR were unsatisfactory compliance and multiple episodes of AR (p = 0.002 each). Urinary infections (n = 13), septicemia (4), tuberculosis (4), CMV disease (7), viral hepatitis (7), and pneumonia (3) were important causes of morbidity. Two patients each had lymphoproliferative disease and new-onset diabetes. There were eight (17.8%) graft losses and six (14%) deaths. The one-, five- and 10-yr graft survivals were 91.1%, 80.4% and 75.1%, respectively; the mean graft survival was 119.4 ± 8.38 months. The respective patient survivals were 95.3%, 87.9%, and 76.9% at one-, five- and 10 yr. Our results affirm that despite scarcity of resources and frequent infections, long-term outcomes of pediatric renal transplantation are highly satisfactory.

Asian Indian donor marrow registry: All India Institute of Medical Sciences experience.

A major limitation in hematopoietic stem cell transplantation (HSCT) is the availability of a genetically matched donor, particularly with respect to the human leukocyte antigens (HLA)-linked immune response genes located on chromosome 6 in humans. During the last 5 years, a total of 688 patients requiring HSCT underwent HLA testing in our department to identify a matched donor from their families. The sibship size ranged from 1 to > or =5 in all disease categories, except thalassemia major where the majority of patients had only 1 sibling. Family genotype analysis revealed that 39.3% of the total number of patients had an HLA-matched sibling and that families with sibship size of > or =4 had a higher probability (68.8%) compared with those with sibship size of < or =3 (29.7%). Because the Indian population is characterized by the presence of novel HLA alleles and unique haplotypes (HLA-A*0211, B*2707, A*26-B*08-DRB1*03), patients with rare HLA alleles have much less probability of finding an unrelated optimally matched donor than those with common HLA phenotypes. Smaller family size and unique HLA profile are limitations that can be overcome by developing unrelated volunteer marrow donor registries. The Asian Indian Donor Marrow Registry at our institute is regularly providing services to such patients.

Frequency distribution of cytokine gene polymorphisms in the healthy North Indian population.

The allelic and genotype frequencies corresponding to 22 single nucleotide polymorphisms (SNPs) in 13 cytokine genes interleukin (IL) 1-alpha (T/C -889), IL1-beta (C/T -511, T/C +3962), IL12 (C/A -1188), interferon-gamma (A/T UTR 5644), transforming growth factor-beta (C/T codon 10, G/C codon 25), tumour necrosis factor-alpha (G/A -308, G/A -238), IL2 (T/G -330, G/T +160), IL4 (T/G -1098, T/C -590, T/C -33), IL6 (G/C -174, G/A nt 565), IL10 (G/A -1082, C/T -819, C/A -592), IL1R (C/T pst11970), IL1RA (T/C mspa111100) and IL4RA (G/A +1902) were determined in 130 healthy North Indian subjects. All genomic typings were performed with polymerase chain reaction with sequence-specific assays. An analysis of the allelic and haplotype frequencies in the North Indian population showed a good fit with the Hardy-Weinberg equilibrium for most of the SNPs. The data can be used for anthropological comparisons, as well as for association studies with different diseases and for use in transplant situations involving acute and chronic rejection.

Immunogenetic association and thyroid autoantibodies in juvenile autoimmune thyroiditis in North India.

OBJECTIVES: Autoimmune thyroid diseases (AITD) encompass a number of conditions that have in common cellular and humoral responses targeting the thyroid gland. Interactions between susceptibility genes and environmental triggers are thought to initiate an autoimmune response to thyroid antigens leading to disease manifestation. Commencement of the disease in childhood leads to the presumption that genetics may have an important role in the causation of the disease. DESIGN: The present study was aimed at evaluating the human leucocyte antigen (HLA) encoded susceptibility to develop juvenile autoimmune thyroiditis (JAT) in patients from North India. PATIENTS: We studied 48 consecutive patients of JAT along with 176 first-degree relatives for their thyroid function (FT4, TSH) and anti-thyroid peroxidase antibody status (AbTPO). MEASUREMENTS: HLA studies were carried out using serology for HLA-class I antigens and DNA analysis of HLA-class II alleles. The data were compared with a cohort of 308 ethnically matched healthy individuals. RESULTS: We observed overt hypothyroidism in 50% and AbTPO positivity in 70.8% of the index cases. Among the first-degree relatives, goitre was observed in 51.7%, thyroid dysfunction in 28.4% and AbTPO in 29.5% of individuals. Of the 37 relatives who underwent fine-needle aspiration cytology (FNAC), 60% had evidence of chronic lymphocytic thyroiditis (CLT). A strong positive association of HLA-DRB1*1404 was observed with the JAT (35.4%vs. 10.4%, chi2 = 19.8, Pc = 0.0001). We also observed a higher (72%, P = 0.03) paternal transmission of HLA-DRB1*1404 to affected offspring in comparison to unaffected offspring. HLA-DRB1*03 was also increased among JAT patients but did not reach statistical significance. CONCLUSION: These studies point towards an important role of immune modifying genes, such as HLA, in influencing susceptibility to juvenile-onset AITD.

Fludarabine, cyclophosphamide and horse antithymocyte globulin conditioning regimen for allogeneic peripheral blood stem cell transplantation performed in non-HEPA filter rooms for multiply transfused patients with severe aplastic anemia.

Multiply transfused patients of severe aplastic anemia are at increased risk of graft rejection. Five such patients underwent peripheral blood stem cell transplantation from HLA-identical siblings with a fludarabine-based protocol. The conditioning consisted of fludarabine 30 mg/m(2)/day x 6 days, cyclophosphamide 60 mg/kg/day x 2 days and horse antithymocyte globulin (ATG) x 4 days. Two different ATG preparations were used: ATGAM (dose 30 mg/kg/day x 4 days) or Thymogam (dose 40 mg/kg/day x 4 days). Engraftment: median time to absolute neutrophil count (ANC) >0.5 x 10(9)/l was 11 days (range: 8-17) and median time to platelet count >20 x 10(9)/l was 11 days (range: 9-17). At a median follow-up of 171 days (range: 47-389), there has been no graft rejection and all patients are in complete remission. Acute GVHD (grade 1) occurred in one patient only. Chronic GVHD developed in two patients (extensive in one and limited in another). The transplants were performed in non-HEPA filter rooms. In only one patient, systemic antifungal therapy (voriconazole) was used. The use of Thymogam brand of ATG for conditioning is being reported for the first time. Our experience suggests that this fludarabine-based protocol allows rapid sustained engraftment in high-risk patients without significant immediate toxicity.

Reconstitution of naïve T cells and type 1 function after autologous peripheral stem cell transplantation: impact on the relapse of original cancer.

BACKGROUND: Phenotypic and functional reconstitution of T cells after peripheral blood stem cell transplantation (PBSCT) and its influence on posttransplant immune status is important in terms of immune surveillance and relapse of original cancer. We investigated the relationship between the dominant immune reconstitution pathway and the immune surveillance. We also tested the cytokine bias acquired by T cells after transplantation and its possible influence on relapse of original malignancy. METHODS: Immunophenotyping of naïve and memory T cells was performed by flow cytometry on patients who underwent PBSCT for various cancers. Cytokine production by peripheral memory helper (CD4) and cytotoxic (CD8) T cells was investigated at various pretransplant and posttransplant time points with fluorescein isothiocyanate-based intracellular cytokine assay after short-term in vitro mitogenic stimulation (phorbol myristate acetate + ionomycin). Data on T-cell subsets and polarized cytokines gamma-interferon (Ifn) and interleukin 4 produced by memory T cells were compared with that of healthy controls. RESULTS: The reconstitution of naïve T cells and gamma-Ifn-producing memory cells was significantly lower in patients who experienced relapse of original cancer within 1 year of PBSCT compared to those who showed no signs of relapse even after 2 years and compared to normal subjects. The results indicate that efficient reconstitution of naïve T cells and type 1 function of memory T cells are important in maintaining T-cell repertoire diversity after PBSCT. It also confers appropriate levels of immune surveillance against diverse neoantigens that evolve from residual tumor burden. The data reveal that chemotherapy-induced thymic injuries may impair regeneration of naïve cells that result in a naivopenic state in a susceptible host. CONCLUSIONS: The study highlights the importance of naïve T-cell reconstitution and points towards cell replacement strategies for improving immune surveillance after PBSCT.

HLA-B27 alone rather than B27-related class I haplotypes contributes to ankylosing spondylitis susceptibility.

Characterization of non-B27 susceptibility genes will be required to know the pathogenesis of AS. The aim of this study was to examine whether ankylosing spondylitis (AS) susceptibility is controlled by B27 alone rather than B27 haplotypes and, whether other closely related class I loci, such as MICA and TNFA genes might play a role in AS. Three hundred eleven B27-positive samples from Caucasoid, Asian, and African populations were selected and genotypes were carried out by PCR/SSOP (HLA-B27 and HLA-C), PCR/SSP (MICA-TM polymorphism in the transmembrane region), PCR/SSCP (MICA alleles), and PCR-RFLP (TNF-alpha). Of these, 161 were AS patients, chosen in order to investigate the contribution of TNFA and MICA loci to AS in HLA-B27 positive individuals. Some findings can be concluded from the study: (a) No significant differences of TNF-alpha promoter alleles at position -308 and -238 (A/G) were found between AS patients and B27 matched alleles from healthy controls; (b) strong linkage disequilibrium was found between the B27 and the MICA alleles. The MICA-A4 was found to be in association with B*2705,02,03 and 08; MICA-A5 with B*2704 and B*2707 and MICA-A.5.1 with B*2706; (c) no significant differences of MICA alleles were found between AS and controls carrying the B27-associated alleles, and therefore no evidence is provided for an additional role of MICA gene in AS susceptibility; (d) there are a striking correlations between the structure of B27 extended haplotypes (from MICA region to HLA-C) and the ethnic distribution of these subtypes. The results of differential linkage disequilibrium with HLA-B27 subtypes suggest that B27 itself remains the primary gene for AS susceptibility, and TNFA and MICA are not involved in the pathogenesis of the disease.

Immunogenetics of peripheral arteriopathies.

Takayasu's arteritis (TA) and thrombangiitis obliterans (Buerger's disease) are idiopathic, inflammatory arteriopathies with strong indications for the involvement of autoimmunity and host genetic factors in their immunopathogenesis. The exact etiology of these arteriopathies still remains unknown even after almost nine decades of their description. A series of immunogenetic studies conducted worldwide seeking to define genetic factors in governing immune response in these diseases have yielded conflicting results on the involvement of HLA molecules. While an association of HLA-B5 or its molecular subtypes with Takayasu's arteriitis has been emphasized in patients from Japan, Korea and India, no such association has been reported in Mexican and North American patients. On the other hand, a limited data is available on the association of HLA antigens with Buerger's disease. In this article, we provide an overview of the immunogenetics of Buerger's disease and Takayasu's arteriitis in the context of studies in North Indian patients and those in other ethnic groups. Our studies indicate a positive association of Takayasu's arteriitis with the HLA-B5 molecule with no preferential association with its two major subtypes. In Buerger's disease, we have observed a strong positive association with HLA-DRB1*1501 consistent with the findings in Japanese patients. These results suggest an important role of HLA linked factors in governing susceptibility to both arteriopathies.

Family studies and human leukocyte antigen class II typing in Indian probands with seizures in association with single small enhancing computed tomography lesions.

PURPOSE: To define the clinical features of the syndrome of seizures associated with single, small, enhancing computed tomography (CT) lesions (SSELs) in 235 Indian probands and seizure types among their family members. Human leukocyte antigen (HLA) class II genomic typing in randomly selected 41 probands was done to identify the role of hereditary factors in this syndrome. METHODS: The seizure types among 235 probands, their clinical outcome, and seizures in their family members were studied. Family data were collected on relatives of 212 additional probands with neurologic diseases other than epilepsy. HLA class II antigens were studied by using polymerase chain reaction (PCR) amplified DNA and sequence-specific oligonucleotide probe (PCR-SSOP) hybridization. RESULTS: The seizures in 86% were partial with or without generalization; 77% had fewer than five seizures before the first CT scan. Evanescent focal neurologic deficits after seizures were noted in 40%. Most patients (97%) were treated with a single antiepileptic drug (AED). Significant resolution of the CT scan lesion was noted within 6 months in 125 (53%) of 235 cases. Two thirds of patients had no seizures while taking a single AED, and an additional 18% had no seizures even after their AEDs were discontinued. Epilepsy among relatives of Indian probands having seizures in association with SSELs was more common as compared with relatives of probands with other neurologic diseases. A family history of seizures was noted in 21% probands, the ratio of affected first- to second-degree relatives was 4.3:1, and 60% of affected sibs had syndromic concordance with probands. There was a positive association of HLA-DRB1*13 (Pc = 0.036) with this syndrome. CONCLUSIONS: The syndrome of seizures in association with SSELs seems to be a benign localization-related epileptic syndrome. Our results of HLA studies point to an inherited susceptibility to an infective agent, which in most cases is of cysticercal etiology.

Treatment of acute rejection in live related renal allograft recipients: a comparison of three different protocols.

We present our experience on the comparison of three different modes of steroid therapy, oral prednisolone (OP), intravenous dexamethasone (IVDX) and intravenous methylprednisolone (IVMP) in the treatment of acute rejection (AR) in renal allograft recipients. Between January 1980 and January 1992, 206 patients underwent live related renal transplantation. Before 1990, all received prednisolone (PRED) and azathioprine (AZA) only. After 1990, patients were given PRED, AZA and cyclosporine (CsA). After 1 year, CsA was stopped and patients were converted to a two-drug regimen only. Of the 206 patients, 180 (87.4%) were male and mean age was 30.3+/-8.7 years (range 14-63). During the mean follow-up of 43.5 months, 178 episodes of AR were seen in 121 patients. Each episode was considered as a separate entrant in the study. Conventional immunosuppression was given in 151 episodes and 27 episodes were on triple-drug therapy. Diagnosis of AR was made by clinical, sonography, nuclear scan with or without graft biopsy evidence. Of the 178 AR, 110 (61.8%) were within 3 months, 36 (20.2%) were between 3 months and 1 year and 32 (18%) were after 1 year. OP was given in 11 cases while IVDX and IVMP were given in 48 and 119 cases respectively. Overall, 154 (86%) showed either a complete or partial response to antirejection therapy. Response to therapy was 91, 90 and 85% in OP, IVDX and IVMP groups respectively. There was no statistical difference in response rate in different groups. There was also no difference in side effects in three different groups. Our data suggest that it is the high dose of steroid rather than mode of therapy which is responsible for therapeutic benefit in treatment of AR.

IMMUNOLOGICAL MONITORING IN TRANSPLANTATION.

Sixty eight patients who had undergone live related donor renal transplantation (LRD), were evaluated for soluble interleukin-2 receptors (sIL-2R), tumour necrosis factor alpha (TNF-α) and autoantibodies against IgG(Fab')2 and IgG(Fc), at pre- and various post-transplant intervals. Serum sIL-2R levels were significantly elevated in hemodialysed patients awaiting transplantation (mean 259.2 ± 90.5 pmol/L) as compared to healthy volunteers (mean 52.6 ± 16.7 pmol/L). In 96 samples obtained from patients with well-functioning grafts (WFG), the post-transplant sIL-2R levels (135.6 ± 65.4 pmol/L) were significantly lower (p < 0.001) than their pretransplant values. Eight patients with cyclosporin-A (CsA) nephrotoxicity, 14 with reversible acute tubular necrosis (ATN) and 4 patients with partial surgical obstruction, revealed moderate levels (99.0 ± 13.7, 184.1 ± 47.5, 156.7 ± 40.4 pmol/L respectively). On the other hand, 29 patients with acute rejection episodes, 11 with chronic rejection and 8 with infections had significantly higher levels (307.9 ± 89.3, 253.3 ± 68.6,345 ± 110.6 pmol/L), (p < 0.001). TNF-α levels were also raised in rejection and infective episodes but were not statistically significant. Serum anti-IgG(Fab')2 levels were found higher (0.407 OD) in WFG as compared to those with declining graft functions (0.279 OD). On the contrary high pre- and post-transplant anti-IgG(Fc) activity was associated with increased graft rejection and lower survival rate.

Occurrence of autoimmune diseases and relationship of autoantibody expression with HLA phenotypes in multicase rheumatoid arthritis families.

Presence of autoimmune diseases and relationship of autoantibody expression with HLA association has been studied in 44 multicase rheumatoid arthritis (RA) families of Asian Indian origin. An increased prevalence of systemic lupus erythematosus (SLE) was observed in relatives (2.3%). Although HLA-DR4 segregated preferentially with seropositivity in general, no difference was observed among seropositive versus seronegative RA. On the other hand, no HLA association was observed with ANF positivity in these families. An increased frequency of DR7 in the ANF negative and RF negative group of RA patients compared to positive groups suggests that it may act as protective element for the development of autoantibodies in RA. An increased occurrence of DR4 in relatives affected with SLE was observed. While RA segregated mostly with HLA-DR4 in these families, autoimmune thyroid disease and insulin dependent diabetes mellitus (IDDM) segregated with HLA-DR3 suggesting the involvement of at least two sets of HLA-linked autoimmunity favouring susceptibility genes in the Indian population.

Is solitary rectal ulcer a manifestation of a systemic disease?

In a prospective study of 22 patients with solitary rectal ulcer, we tried to define the features of this condition, especially the associated systemic features, that may give some clues to its etiopathogenesis. In 15 of these patients a single rectal ulcer was found, whereas in seven patients two ulcers were present in each. Of the total 29 ulcers in these patients, 19 were located on the anterior or anterolateral wall of the rectum and 10 were on the posterior or posterolateral wall. The sigmoidoscopic appearance of the ulcer was quite characteristic, yet a biopsy was considered essential to rule out other pathologic processes. Histological features of the solitary rectal ulcer comprised fibrous obliteration of the lamina propria with disorientation of the muscularis mucosa and extension of muscle fibers into the lamina propria. Evident rectal prolapse was present in only three patients. Recurrent oral ulcerations occurred in four (18.2%) patients and erythema nodosum in one of them (4.5%). Sacroiliitis was present in six of 19 (31%) patients studied radiologically, and human leukocyte antigen (HLA)-B27 occurred in four of the 20 patients (20%) tested for HLA class I antigens. All the four HLA-B27-positive patients had associated sacroiliitis and showed good response to sulfasalazine. These associations raise the possibility that solitary rectal ulcers may be a part of a systemic disease or of several diseases with varied etiology.

Imbalances in T cell subpopulations in human gliomas.

The quantitation of cells bearing CD3, CD4, CD8, and B cell phenotypic markers, as well as an estimation of serum immunoglobulin (Ig)G, IgA, and IgM, was carried out in a group of 39 glioma patients with different grades of malignancy. The findings were compared with those obtained from 21 normal healthy control subjects. The analysis revealed a significant decrease both in the absolute numbers and in the percentages of circulating CD3+ (p less than 0.001) and CD4+ (p less than 0.001) cells, while the CD8+ and Pan B+ cells remained within the normal range irrespective of the type and grade of tumor. The CD4+:CD8+ ratio was significantly decreased in all categories of patients. The CD4 lymphopenia was also evident in 10 patients who had no history of previous immunosuppressive drug therapy (steroids and anticonvulsants) until the commencement of the study. The Ig levels were within the normal range in patients with malignant astrocytoma and glioblastoma multiforme, whereas a three- and fourfold increase in the IgM level was observed in patients with astrocytoma. It is suggested that T cell lymphopenia in glioma patients could mainly be due to a selective depletion of CD4+ cells and that it occurs principally as a reaction to the tumor.

HLA status following fetal liver transplantation in aplastic anaemia and acute myeloid leukaemia.

Fetal liver infusion (FLI) was tried as an alternate mode of therapy in 40 patients with aplastic anaemia and in 16 patients with acute myeloid leukaemia. The fetal HLA typing carried out on spleen and thymus cells revealed that, while it was more difficult to HLA type the thymus than the spleen cells, 'full house' antigens could be determined only in fetuses of 18 weeks or older. No special effort was made to transfuse HLA- matched or partially matched donor cells into the recipient. The recipients were HLA typed at varying time intervals following FLI in an attempt to document a possible chimerism. None of the patients revealed a 'shift' in their HLA antigen profile and there was no evidence of any donor cell engraftment. No relationship between the HLA match of donor and recipient, and the general condition, the prognosis or the total survival of the patient was evidenced. These data indicate that, even though fetal liver cells express HLA antigens, these cells are functionally incompetent to cause an apparent graft-versus-host disease in the host.

Studies on engraftment following fetal liver infusion.

Studies to find engraftment following fetal liver infusion (FLI) in aplastic anaemia (AA) and acute myeloid leukaemia (AML) were carried out in 24 patients (17 AA and 7 AML patients) out of the 56 who received FLI. HLA studies done in 13 patients (3 AA and 5 AML), repeatedly after FLI, showed no significant change in HLA antigen pattern before and after FLI. Red cell antigen studies were done in five (1 AA and 4 AML) patients, 3 weeks to 7 months after FLI. One patient with AML who was Rh negative prior to reinduction chemotherapy became Rh positive two months after FLI; six months later he was Rh negative again. In the remaining patients there was no change in red cell antigen pattern after FLI. Radio-immuno-assay to detect alpha-fetoprotein levels, carried out in 10 (8 AA and 2 AML) patients repeatedly after FLI, demonstrated no increase. In 13 patients (8 AA and 5 AML) in whom there was a sex difference between donor and recipient, bone marrow cultures for sex chromosomes revealed mixture of XX and XY cells in 3 male patients with aplastic anaemia. One male patient with AML demonstrated complete engraftment after induction chemotherapy and FLI: all the mitoses studied were of XX pattern. Engraftment was however temporary as repeated studies revealed reversion to XY pattern. The present work suggests that infusion of fetal liver cells may sometimes induce temporary chimerism or engraftment in an adult host; in the majority of cases, however, engraftment could not be established.