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Objectives Immune factors determining clinical progression following HIV-1 infection remain unclear. The SPARTAC trial randomized 366 participants in primary HIV infection (PHI) to different short-course therapies. The aim of this study was to investigate how early immune responses in PHI impacted clinical progression in SPARTAC. Design and methods Participants with PHI recruited to the SPARTAC trial were sampled at enrolment, prior to commencing any therapy. HIV-1-specific CD4(+) and CD8(+) ELISpot responses were measured by gamma interferon ELISPOT. Immunological data were associated with baseline covariates and times to clinical progression using logistic regression, Kaplan-Meier plots, and Cox models. Results Making a CD4(+) T-cell ELISpot response (n = 119) at enrolment was associated with higher CD4(+) cell counts (P = 0.02) and to some extent lower plasma HIV RNA (P = 0.07). There was no correlation between the number of overlapping Gag CD8(+) T-cell ELISpot responses (n = 138) and plasma HIV-1 RNA viral load. Over a median follow-up of 2.9 years, baseline CD4(+) cell ELISpot responses (n = 119) were associated with slower clinical progression (P = 0.01; log-rank). Over a median of 3.1 years, there was no evidence for a survival advantage imposed by CD8(+) T-cell immunity (P = 0.82). Conclusion These data support a dominant protective role for CD4(+) T-cell immunity in PHI compared with CD8(+) T-cell responses, and are highly pertinent to HIV pathogenesis and vaccines, indicating that vaccine-induced CD4(+) responses may confer sustained benefit.

OBJECTIVE: To determine the cause of hyperglycemia in childhood diarrhea. METHODS: During an 8-month period, patients admitted to a diarrhea treatment center in Bangladesh had their blood glucose concentrations determined. Sixteen patients aged 2 to 10 years with hyperglycemia (blood glucose concentration >10.0 mmol/L) and 20 patients in the same age group with a normal blood glucose concentration (3.3 to 9.0 mmol/L) had blood samples obtained on admission and 4 and 24 hours later for determination of glucoregulatory hormones and gluconeogenic substrates. RESULTS: Prevalence of hyperglycemia among patients aged 2 to 10 years was 9.4%. Compared with the normoglycemic patients, hyperglycemic patients more often had severe dehydration (100% versus 10%, p

OBJECTIVE: A minority of HIV-1 positive individuals treated with antiretroviral therapy (ART) in primary HIV-1 infection (PHI) maintain viral suppression on stopping. Whether this is related to ART duration has not been explored. DESIGN: And Methods: Using SPARTAC trial data from individuals recruited within 6 months of seroconversion, we present an observational analysis investigating whether duration of ART was associated with post-treatment viraemic control. Kaplan-Meier estimates, logistic regression and Cox models were used. RESULTS: 165 participants reached plasma viral loads (VL) 12 weeks compared to ≤12 weeks (p=0.061). Cumulative probabilities of remaining 12 weeks. In multivariable regression, ART for >12 weeks was independently associated with a lower probability of being ≥400 copies/ml within 12 weeks of ART stop (OR=0.11 (95%CI=0.03,0.34), p

Hepatitis C virus (HCV) sets up persistent infection in the majority of those exposed. It is likely that, as with other persistent viral infections, the efficacy of T-lymphocyte responses influences long-term outcome. However, little is known about the functional capacity of HCV-specific T-lymphocyte responses induced after acute infection. We investigated this by using major histocompatibility complex class I-peptide tetrameric complexes (tetramers), which allow direct detection of specific CD8+T lymphocytes ex vivo, independently of function. Here we show that, early after infection, virus-specific CD8+T lymphocytes detected with a panel of four such tetramers are abnormal in terms of their synthesis of antiviral cytokines and lytic activity. Furthermore, this phenotype is commonly maintained long term, since large sustained populations of HCV-specific CD8+T lymphocytes were identified, which consistently had very poor antiviral cytokine responses as measured in vitro. Overall, HCV-specific CD8+T lymphocytes show reduced synthesis of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) after stimulation with either mitogens or peptides, compared to responses to Epstein-Barr virus and/or cytomegalovirus. This behavior of antiviral CD8+T lymphocytes induced after HCV infection may contribute to viral persistence through failure to effectively suppress viral replication.

Human immunodeficiency virus type 1 (HIV-1) evokes a strong immune response, but the virus persists. Polymorphisms within known antigenic sites result in loss of immune recognition and can be positively selected. Amino acid variation outside known HLA class I restricted epitopes can also enable immune escape by interfering with the processing of the optimal peptide antigen. However, the lack of precise rules dictating epitope generation and the enormous genetic diversity of HIV make prediction of processing mutants very difficult. Polymorphism E169D in HIV-1 reverse transcriptase (RT) is significantly associated with HLA-B*0702 in HIV-1-infected individuals. This polymorphism does not map within a known HLA-B*0702 epitope; instead, it is located five residues downstream of a HLA-B*0702-restricted epitope SPAIFQSSM (SM9). Here we investigate the association between E169D and HLA-B*0702 for immune escape via the SM9 epitope. We show that this single amino acid variation prevents the immune recognition of the flanked SM9 epitope by cytotoxic T cells through lack of generation of the epitope, which is a result of aberrant proteasomal cleavage. The E169D polymorphism also maps within and abrogates the recognition of an HLA-A*03-restricted RT epitope MR9. This study highlights the potential for using known statistical associations as indicators for viral escape but also the complexity involved in interpreting the immunological consequences of amino acid changes in HIV sequences.

Cytotoxic T lymphocytes (CTLs) are thought to play a crucial role in the termination of the acute primary HIV-1 syndrome, but clear evidence for this presumption has been lacking. Here we demonstrate positive selection of HIV-1 proviral sequences encoding variants within a CTL epitope in Nef, a gene product critical for viral pathogenicity, during and after seroconversion. These positively selected HIV-1 variants carried epitope sequence changes that either diminished or escaped CTL recognition. Other proviruses had mutations that abolished the Nef epitope altogether. These results provide clear evidence that CTLs exert selection pressure on the viral population in acute HIV-1 infection.

Renal function was monitored in 24 patients with systemic envenoming following proven Russell's viper bite. In all patients, blood clotted within 20 min on admission. In 15 cases severe defibrination (systemic envenoming) developed during the next 3-5 d. None of the patients received antivenom before admission but enzyme-refined monospecific antivenom was given to those who developed signs of systemic envenoming. Specific antigen was detected by enzyme immuno-assay in all 21 subjects tested. Nine patients whose renal function remained normal did not develop systemic envenoming, and recovered without any treatment even though venom antigen was detectable in their serum. Ten patients developed mild renal dysfunction and systemic envenoming, but recovered after treatment with antivenom alone. The remaining 5 patients, all of whom were oliguric from admission, developed acute renal failure despite treatment with antivenom, but some recovered after peritoneal dialysis. Serum venom antigen levels were high in the last 2 groups, but there was some overlap. Albuminuria, found only in patients who became systemically envenomed, was associated with high fractional sodium excretion in those who developed acute oliguric renal failure. Albuminuria may appear before a gross clotting defect is detectable. It is an indication for antivenom and spot measurements might prove a useful early predictor of outcome.

In Anuradhapura, Sri Lanka, 5 patients proved to have been bitten by common kraits (Bungarus caeruelus) and 2 by Sri Lankan cobras (Naja naja naja) were investigated. In all the cases of krait bite the patients were bitten while they were asleep: local signs were negligible but 4 developed symptoms of systemic envenoming including paralysis, muscle pain and tenderness and abdominal pain. Mild myoglobinaemia was found in one case. Of the 2 patients bitten by cobras, one developed severe local swelling which progressed to necrosis and the other local swelling and respiratory paralysis. Response to polyspecific antivenom (Haffkine, India) was neither rapid nor convincing. Venom antigenaemia became undetectable within 2 h of the start of antivenom treatment, but recurred 25 and 65 h later in 2 cases. Among a group of 27 patients treated with this antivenom (including 21 bitten by Russell's vipers), the incidence of early anaphylactic and pyrogenic reactions was high at 52% and 65% respectively. Anticholinesterase did not improve paralysis in 2 patients bitten by kraits. The respiratory failure in 2 patients was successfully treated by mechanical ventilation for 8 and 30 h. These observations confirm the importance of neurotoxic symptoms following bites by these species but also suggest a contributory role of generalized rhabdomyolysis in krait victims and emphasize the problem of severe local tissue necrosis in cobra victims. There is a need for safer and more potent antivenoms for use in Sri Lanka.

Cytotoxic T lymphocytes (CTL) play a central role in containment of HIV infection. Evasion of the immune response by CTL escape is associated with progression to disease. It is therefore hypothesised that transmitted viruses encode escape mutations within epitopes that are required for successful control of viraemia. In order to test this hypothesis, escape through the dominant HLA-A2-restricted CTL epitope SLYNTVATL (p17 Gag residues 77–85 SL9) in the setting of mother-to-child-transmission (MTCT) was investigated. Initial data from two families in which the HIV-infected mother expressed HLA-A*0201 and had transmitted the virus to other family members were consistent with this hypothesis. In addition, analysis of the gag sequence phylogeny in one family demonstrated that CTL escape variants can be successfully transmitted both horizontally and vertically. To test the hypothesis further, a larger cohort of transmitting mothers (n=8) and non-transmitters (n=14) were studied. Variation within the SL9 epitope was associated with expression of HLA-A2 (P=0.04) but overall no clear link between variation from the SL9 consensus sequence and MTCT was established. However, the high level of background diversity within p17 Gag served to obscure any possible association between escape and MTCT. In conclusion, these studies highlighted the obstacles to demonstrating CTL escape arising at this particular epitope. Alternative strategies likely to be more definitive are discussed.

There are significant associations between possession of certain HLA class I alleles and rate of progression to AIDS. Immunological data provide an explanatory mechanism for this relationship. Patients with HLA types associated with rapid disease progression recognize a significantly smaller fraction of their known repertoire of viral epitopes than do patients with HLA types associated with slow progression. Population frequency of HLA types (or supertypes) and their capacity to elicit cytotoxic T lymphocyte responses are also negatively correlated. These data provide an immunological mechanism to explain HLA-related risk of progression to AIDS and emphasize the central role of viral evolution in the pathogenesis of HIV.

Although hepatitis C virus (HCV) infection is very common, identification of patients during acute infection is rare. Consequently, little is known about the immune response during this critical stage of the disease. We analyzed the T lymphocyte response during and after acute resolving HCV infection in three persons, using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and human histocompatibility leukocyte antigen (HLA) peptide tetramer assays. Acute infection was associated with a broadly directed T helper and cytotoxic T lymphocyte (CTL) response, which persisted after resolution of clinical hepatitis and clearance of viremia. At the earliest time point studied, highly activated CTL populations were observed that temporarily failed to secrete IFN-γ, a “stunned” phenotype, from which they recovered as viremia declined. In long-term HCV-seropositive persons, CTL responses were more common in persons who had cleared viremia compared with those with persistent viremia, although the frequencies of HCV-specific CTLs were lower than those found in persons during and after resolution of acute HCV infection. These studies demonstrate a strong and persistent CTL response in resolving acute HCV infection, and provide rationale to explore immune augmentation as a therapeutic intervention in chronic HCV infection.

Proliferative responses of T lymphocytes to antigens specific and not specific for malaria were investigated in 32 adult patients in eastern Thailand during acute infection with Plasmodium falciparum malaria and during their convalescence. Immune unresponsiveness to malarial antigen, which persisted for more than four weeks in 37.5% of the individuals, was present in all patients, irrespective of parasitemia or severity of clinical illness. Suppression of responses to nonspecific antigens was less profound and observed only in patients with moderately severe or cerebral malaria. The depressed functional responses were associated with a loss of T lymphocytes--both helper and suppressor subsets--from the peripheral blood; these responses were recovered once parasites were cleared. These results indicate that blood-stage plasmodial infections may suppress responses important for immunity to malaria and so allow the parasite to survive. They further suggest that patients acutely or even recently infected with P. falciparum may not respond as well to a malaria vaccine as would uninfected individuals.

Patients who are severely envenomed by Russell's viper develop DIC which is frequently associated with spontaneous bleeding and incoagulable blood. These haemostatic disturbances may be responsible for death or organ/tissue damage both through haemorrhage and microvascular occlusion by fibrin thrombi. The most striking laboratory features of the coagulopathy developing after Russell's viper bite in the 42 patients studied were depletion of fibrinogen (mean 0.09 g/l, range 0-0.6), factor V (6.5 u/dl, range 0-17), factor X (35 u/dl, range 1-85), factor XIIIa (57 u/dl, range 15-82), plasminogen (61 u/dl, range 10-92), antiplasmin (36 u/dl, range 14-62). Protein C (49 u/dl, range 15-100) and platelets (104 x 10(9)/l, range 25-197). Intense fibrinolytic activity was detected in all cases with marked elevation of FDPs (1614 micrograms/ml, range 350-3000), a large proportion of which were cross-linked (1058 micrograms/ml, range 38-3000). The monospecific Burmese antivenom appeared to be very effective in neutralizing the venom procoagulants and in restoring blood coagulability. Moreover, the unexpectedly normal level of AT III provides a theoretical basis for the use of heparin to enhance the inactivation of those serine proteases present before antivenom administration.

Erythrocyte survival times were measured in healthy Thai controls and in patients following clearance of asexual P. falciparum or P. vivax parasitaemia. In five controls the mean cell life (MCL) of compatible donor erythrocytes was 89.6 d (mean range 73-101 d) compared with a mean MCL of 56.8 d (range 30-66 d) for autologous erythrocytes in 12 falciparum patients. In one of these patients the survival curve was biphasic with a rapid loss of some labelled cells. The survival of compatible donor erythrocytes was also studied in 10 patients and two types of survival curve could be distinguished. In five patients the cells had a mean MCL of 64.4 d (range 42-90 d). In the others survival curves were curvilinear, suggesting a complex mechanism of cell clearance or the presence of more than one cell population. There was initially a more rapid rate of destruction. In P. vivax malaria the MCL of autologous erythrocytes in seven patients was a mean of 67.2 d (range 34-74 d) and that of compatible donor cells in six patients was 66.8 d (range 54-76 d). In all except one of these patients both autologous and donor cell survival curves could be fitted to straight lines. No increase in cell-bound IgG or C3 was evident in 12 patients tested. The differences between the mean MCL in all the groups of patients and the controls were statistically significant at the 5% level. This indicates an increased rate of erythrocyte destruction following clearance of P. falciparum or P. vivax parasites which is not antibody or complement mediated. The mechanism is unknown, but appears to be extrinsic to the erythrocytes themselves and may result from nonspecific activation of the reticuloendothelial function associated with the parasitic infection.

Of 123 patients with proved Russell's viper bite, 28% showed no evidence of envenoming, 28% had local swelling alone, but 44% had systemic envenoming manifested by incoagulable blood (100% of those admitted before treatment), thrombocytopenia (26%), spontaneous systemic bleeding (20%), hypotension (35%), evidence of increased capillary permeability (24%), and oliguria (44%). Patients with systemic envenoming usually had more local swelling than those without, but 5 had no local signs. Snake length correlated with the amount of local swelling, but snakes causing systemic envenoming were no longer than those causing local or no envenoming. Burma Pharmaceutical Industry monospecific antivenom was rapidly effective in restoring blood coagulability but did not prevent the development of renal failure even when given within 4 h of the bite. Hypotension responded to volume expanders (11/19 cases) and dopamine (6/7 cases) but not to naloxone (0/3) or high-dose methylprednisolone (0/5). The 10 deaths (8%) were attributed to hypotension, pituitary haemorrhage, and renal failure.

BACKGROUND: Virus-specific cellular immune responses mediated by CD4 and CD8 T lymphocytes are thought to be central to the effective control of HIV-1 replication in vivo. However, quantitative correlations between HIV-specific T lymphocyte frequencies and plasma virus load (pVL) have proved difficult to establish in infected human individuals. This most likely reflects the complex interactions between the virus and these immune effector cells in the absence of treatment. OBJECTIVE: To assess frequencies of HIV-specific T lymphocytes after prolonged suppression of viral replication, i.e., under conditions where the effects of virus on the immune response are standardized and minimized, thereby fixing an important variable in a dynamic multivariate system. METHODS: HIV-specific CD4 and CD8 T lymphocyte frequencies were measured in 122 individuals after prolonged periods of successful combination antiretroviral therapy (ART) administered during chronic HIV-1 infection. RESULTS: The residual frequency of both CD4 and CD8 T lymphocytes specific for HIV-1 was inversely related to the pretreatment pVL. This relationship appeared to be non-linear, indicating the presence of a threshold pretreatment pVL level above which HIV-specific CD4 and CD8 T lymphocyte responses could not be maintained when antigenic drive was suppressed. Substantial populations of functional HIV-specific CD4 and CD8 T lymphocytes were generally detectable after prolonged ART only in those individuals with a pretreatment plasma HIV-1 RNA < 100,000 copies/ml. CONCLUSION: These findings identify a quantitative immune associate of host-virus interactions in established HIV-1 infection.

Amodiaquine hydrochloride (3 mg base kg-1) was given by constant rate intravenous injection over 10 min to seven healthy adult male volunteers, and by constant rate infusion (10 mg base kg-1) over 4 h to 10 adult patients admitted to hospital with falciparum malaria. After intravenous injection in volunteers there was considerable variation in plasma concentration profiles between subjects; peak plasma concentrations ranged between 65 and 1921 ng ml-1. A biexponential equation was fitted to the plasma concentration time data and the following estimated pharmacokinetic parameters (geometric mean; range) were derived; lambda 1 = 24.4 (7.6-95.0) h-1, lambda 2 = 0.33 (0.12-0.79) h-1, V1:1.1 (0.3-3.6) 1 kg-1, Vss: 17.4 (2.3-95.9) 1 kg-1 and systemic clearance 13.0 (4.7-56.6) 1 kg-1 h-1. After intravenous infusion there was also considerable variability between patients with post-infusion plasma concentrations ranging between 82 and 836 ng ml-1. The plasma concentration-time profiles were biphasic with the following estimated pharmacokinetic parameters (geometric mean; range) alpha = 1.87 (0.60-8.52) h-1, beta = 0.069 (0.021-0.265) h-1, V1: 4.6 (0.5-29.3) 1 kg-1, Vss: 38.3 (3.7-127.9) 1 kg-1 and systemic clearance CL (1.6-17.3) 1 kg-1 h-1. There was no measurable long terminal elimination phase, and the principal metabolite desethyl amodiaquine was not detected in the plasma samples. There was no serious toxicity in either group. During intravenous injection there was a significant fall in systolic blood pressure in four subjects (mean fall 16 mm Hg) but there was no significant change in heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Chloroquine diphosphate (15 mg base kg-1) was given by constant rate intravenous infusion to two groups of Thai subjects. Eleven were patients with malaria (10 with Plasmodium vivax and one case with Plasmodium malariae) and 10 were healthy normal volunteers. 2. Plasma and packed red-cell concentrations of chloroquine, electrocardiographic intervals, arterial blood pressure and pulse were measured at frequent intervals. 3. Peak plasma concentrations at the end of the infusion ranged from 979 to 2,900 ng ml-1 in the malaria patients. In the group of healthy subjects the range was 550-2,200 ng ml-1. Values for terminal elimination rate constant, (lambda z) plasma clearance (CL), initial volume of distribution (V1) and volume of distribution at steady state (Vss) were calculated. For the healthy subjects, mean estimates of these parameters were lambda z = 0.062 +/- 0.030 day-1, CL = 597 +/- 238 ml min-1, V1 = 0.66 +/- 0.71 l kg-1 and Vss = 132 +/- 50 l.kg-1 For the group of malaria patients, the corresponding values were lambda z = 0.055 +/- 0.032 day-1, CL = 535 +/- 246 ml min-1, V1 = 0.74 +/- 0.75 l kg-1 and Vss = 136 +/- 64 l kg-1 There was no statistically significant difference in the estimates for any parameter between groups (P less than or equal to 0.05). 4. Chloroquine concentrations in packed red blood cells consistently exceeded those in plasma and showed no consistent change with time throughout the period of study in either group. The median value for the red cell to plasma ratio was between 3 and 4 in each group.(ABSTRACT TRUNCATED AT 250 WORDS)

The evolution of coagulation abnormalities was studied in Russell's viper bite victims who, on admission to hospital, showed no clinical signs of systemic envenoming. Based on the laboratory results and subsequent clinical course, three groups were distinguished. The first group, consisting of five cases, showed no activation of coagulation at any stage. The second group, consisting of six cases, developed mild to moderate abnormalities in some tests, particularly in the aPTT and factor V assay, which corrected to normal without treatment. The third group, consisting of nine patients, developed haemostatic abnormalities as early as 1-2 h after the bite, which progressed to severe defibrination 4-8 h later at which time antivenom was given. Comparison of the haemostatic abnormalities in the three groups suggested that serial monitoring of the serum FDP concentration may be of value in predicting the likelihood of systemic envenoming and progression to complete defibrination.

Bone marrow aspirates from patients with cerebral malaria were studied with the light and electron microscopes. Various abnormalities were found including: (1) an increase in plasma cells and macrophages, sometimes to a marked degree; (2) phagocytosis of parasitized red cells by macrophages and of merozoites by neutrophil metamyelocytes, neutrophil granulocytes and macrophages; (3) an increase in the proportion of eosinophil granulocytes and their precursors; (4) the presence of giant metamyelocytes; and (5) morphological abnormalities of erythroblasts, particularly irregularly-shaped nuclei and karyorrhexis. A high percentage of the red cells within marrow sinusoids were parasitized and the parasitized cells were attached to the endothelium. Some marrow sinusoids were packed with and completely obstructed by parasitized cells. Strands of electron-dense material were sometimes found connecting the knobs of parasitized red cells to endothelial cells or to the knobs of adjacent parasitized red cells. A striking finding was a complex interdigitation between cytoplasmic processes developed by some of the parasitized red cells and those developed by the endothelial cells to which they were attached. Occasionally, cytoplasmic processes arising from marginated parasitized red cells completely penetrated the endothelial cell and emerged extravascularly. Several parasitized red cells were also found extravascularly between haemopoietic cells. Sequestration of parasitized red cells within small blood vessels may play a part in the pathogenesis not only of the encephalopathy of cerebral malaria but also of the bone marrow dysfunction in severe malaria.

Recent studies using MHC class I tetramers have shown that CD8+ T cell responses against different persistent viruses vary considerably in magnitude and phenotype. At one extreme, hepatitis C virus (HCV)-specific CD8+ T cell responses in blood are generally weak and have a phenotype that is perforin low and CCR7 high (early memory). At the other, specific responses to CMV are strong, perforin high, and CCR7 low (mature or effector memory). To examine the potential mechanisms behind this diversity, we compared CMV-specific responses in HCV-infected and healthy individuals. We find a striking difference in the phenotype of CMV-specific CD8+ T cells between these groups. In the HCV-infected cohort, CMV-specific CD8+ T cells lost markers associated with maturity; they had increased expression of CCR7 and reduced expression of Fas and perforin. They nevertheless responded to Ag in vitro in a manner similar to controls, with strong proliferation and appropriate acquisition of effector memory markers. The reduction in mature CD8 T cells in HCV-infected individuals may arise through either impairment or regulation of T cell stimulation, or through the early loss of mature T cells. Whatever the mechanism, HCV has a pervasive influence on the circulating CD8+ T cell population, a novel feature that may be a hallmark of this infection.

Hepatitis C virus (HCV) readily sets up a persistent infection and is a major cause of liver disease worldwide. Interferon alfa and ribavirin therapy lead to sustained clearance of virus in 31% to 64% of patients with type 1 and non-type 1 genotypes, respectively. It is not clear to what extent these drugs act directly to reduce HCV replication, or indirectly via host immune responses, and what evoked immune responses are associated with clinical outcome. We have examined prospectively 15 patients with chronic HCV infection before, during, and after combination therapy. Quantitative assays for HCV antigen-specific CD4+ and CD8+ T-cell responses, and flow cytometric assays for analysis of the phenotype of T cells, in addition to viral sequencing of core protein, were performed throughout the treatment and follow-up period over 18 months. We found enhancement of proliferative T-cell responses during therapy. Proliferative responses are strikingly heterogeneous in terms of specificity, kinetics, and magnitude. Proliferative responses are often not associated with interferon-gamma release. T-cell responses are rarely sustained irrespective of treatment outcome and this is not due to the evolution of new immune escape variants. T-cell responses tend to peak late in the course of treatment. In conclusion, combination therapy for HCV has a transient effect on host virus-specific T cells in the blood. Induction of sustained T-cell responses may require additional immune modulation later in therapy.

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials.Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20

Hepatitis C virus (HCV) infection is very common worldwide, but has a broad range of outcomes. A minority of patients are able to clear infection spontaneously, and this is thought to be due to the emergence and maintenance of effective cell-mediated immunity, particularly CD4+ T lymphocyte responses. Furthermore, genetic studies have indicated that HLA class II genotype strongly influences the outcome of infection. We have therefore investigated the influence of the protective HLA class II haplotype (DQB1*0301, which is in tight linkage disequilibrium with DRB1*1101) on the CD4+ T lymphocyte responses to HCV. We observe a strong association between this genotype and maintenance of a multispecific CD4+ T helper response. The effect on T helper responses was also maintained after combination interferon-alpha/ribavirin therapy, although the latter influenced the pattern of viral antigens to which patients responded. This is the first disease in which an association of HLA genotype with clinical outcome has been linked to an alteration of the immunological phenotype. The selection of protective peptides in those with the favourable HLA class II genotype may point in the direction of suitable vaccine candidates.

The precise role played by HIV-specific cytotoxic T lymphocytes (CTL) in HIV infection remains controversial. Despite strong CTL responses being generated during the asymptomatic phase, the virus persists and AIDS ultimately develops. It has been argued that the virus is so variable, and the virus turnover so great that escape from CTL recognition would occur continually, but so far there is limited evidence for CTL escape. The opposing argument is that evidence for CTL escape is present but hard to find because multiple anti-HIV immune responses are acting simultaneously during the asymptomatic phase of infection. We describe six donors who make a strong CTL response to an immunodominant HLA-B27-restricted epitope. In the two donors who progressed to AIDS, CTL escape to fixation by the same mutation was observed, but only after 9-12 years of epitope stability. CTL escape may play an important role in the pathogenesis of HIV infection.

HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNFα, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR Vα7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.

Background\ud Short-course antiretroviral therapy (ART) in primary human immunodeficiency virus\ud (HIV) infection may delay disease progression but has not been adequately evaluated.\ud Methods\ud We randomly assigned adults with primary HIV infection to ART for 48 weeks, ART\ud for 12 weeks, or no ART (standard of care), with treatment initiated within 6 months\ud after seroconversion. The primary end point was a CD4+ count of less than 350 cells\ud per cubic millimeter or long-term ART initiation.\ud Results\ud A total of 366 participants (60% men) underwent randomization to 48-week ART\ud (123 participants), 12-week ART (120), or standard care (123), with an average followup\ud of 4.2 years. The primary end point was reached in 50% of the 48-week ART\ud group, as compared with 61% in each of the 12-week ART and standard-care groups.\ud The average hazard ratio was 0.63 (95% confidence interval [CI], 0.45 to 0.90;\ud P = 0.01) for 48-week ART as compared with standard care and was 0.93 (95% CI,\ud 0.67 to 1.29; P = 0.67) for 12-week ART as compared with standard care. The proportion\ud of participants who had a CD4+ count of less than 350 cells per cubic millimeter\ud was 28% in the 48-week ART group, 40% in the 12-week group, and 40% in the\ud standard-care group. Corresponding values for long-term ART initiation were 22%,\ud 21%, and 22%. The median time to the primary end point was 65 weeks (95% CI,\ud 17 to 114) longer with 48-week ART than with standard care. Post hoc analysis\ud identified a trend toward a greater interval between ART initiation and the primary\ud end point the closer that ART was initiated to estimated seroconversion (P = 0.09),\ud and 48-week ART conferred a reduction in the HIV RNA level of 0.44 log10 copies\ud per milliliter (95% CI, 0.25 to 0.64) 36 weeks after the completion of short-course\ud therapy. There were no significant between-group differences in the incidence of the\ud acquired immunodeficiency syndrome, death, or serious adverse events.\ud Conclusions\ud A 48-week course of ART in patients with primary HIV infection delayed disease\ud progression, although not significantly longer than the duration of the treatment.\ud There was no evidence of adverse effects of ART interruption on the clinical outcome.\ud (Funded by the Wellcome Trust; SPARTAC Controlled-Trials.com number,\ud ISRCTN76742797, and EudraCT number, 2004-000446-20.)

Cytotoxic T lymphocytes (CTL) directed against human immunodeficiency virus (HIV)-1 are detectable in the majority of infected individuals, and their early appearance as the initial viremia is suppressed is thought to represent a potent antiviral response. Variation which arises in CTL epitopes can affect recognition by CTL, and we have observed previously that variant epitopes in HIV-1 gag which arise in HIV-1-seropositive donors may act as T cell receptor (TCR) antagonists of their own CTL (Klenerman et al., Nature 1994, 369: 403). The most important question arising from these observations is the extent of these immune escape mechanisms in vivo. Here we show that fresh, uncultured lymphocytes taken directly from HIV-1-infected patients are susceptible to TCR antagonism by variants present within their own virus. In contrast to HLA Class II-restricted T cell responses, where anergy may be induced, we find that in vitro, natural variants may stimulate and sustain growth of CTL. These CTL lines retain lytic specificity exclusively for the original peptide. If this represents events in vivo, natural HIV altered peptide ligands (APL) have the capacity to inhibit the range of CTL directed against an epitope, not simply those clones selected in vitro. Partial activation of CTL by APL could also act to drive an ineffectual CTL response incapable of lysing infected cells bearing these natural antigenic variants. Distortion of lymphocyte populations and function by APL might represent a further mechanism of immune evasion by HIV.

CXCR4-tropic (X4) HIV-1 variants are associated with faster disease progression compared with CCR5-tropic variants; however, the mechanism for this is unclear. We measured T-cell activation in 120 individuals with primary HIV-1 infection. X4-utilizing variants, determined genotypically, were present in 8.3% of the participants and were associated with higher levels of CD4 T-cell activation, even after adjusting for other prognostic factors. Increased CD4 T-cell activation may influence the more rapid immunological decline associated with X4 virus.

The Journal of Infectious Diseases, 202 (10), ISSN:0022-1899, ISSN:1537-6613

Background We aimed to characterize the molecular epidemiology of HIV type-1 (HIV-1) and the prevalence of drug- associated mutations prior to initiating highly active antiretroviral therapy (HAART) in the Free State province, South Africa. The Free State has a population of 3 million, an antenatal HIV prevalence of approximately 34% and a well established infrastucture for antiretroviral (ARV) provision. Methods HIV-1 polymerase genes were sequenced from 425 HAART-naive HIV-1-positive patients at voluntary primary healthcare HIV testing centres, who were subsequently attending district centres for assessment for commencing ARVs. Patients (>18 years) were sampled randomly with no exclusion for gender or clinical criteria. Sequences were analysed according to phylogeny and drug resistance. Results Phylogenetic clustering within the cohort was suggestive of multiple introductions of subtype C virus into the region. Drug resistance mutations (according to the International AIDS Society-USA classification) were distributed randomly across the cohort phylogeny with an overall prevalence of 2.3% in the sampled patients. When stratified according to CD4+ T-cell count, the prevalence of resistance was 3.6%, 0.9% and 1.2% for CD4+ T-cell counts 500 cells/μ l, respectively, and was most common for non-nucleoside reverse transcriptase inhibitor resistance (3.1% in patients with CD4+ T-cell count + T-cell counts ( P=0.003), suggesting unrecognized exposure to ARVs. Conclusions In the Free State population, there was a statistical association between low CD4+ T-cell counts and drug-associated viral polymorphisms. Our data advocate the benefit of detailed history taking from patients starting HAART at low CD4+ T-cell counts with close follow-up of the virological response.

Summary Early anti-retroviral treatment (ART) in primary human immunodeficiency virus (HIV) infection (PHI) may have unique, restorative immunological and virological benefits which could enhance clinical outcomes. However, the sustainability of these HIV-specific immune responses and their impact on clinical outcome remains unclear. We present a 3-year longitudinal clinical and immunological follow-up of a single-arm, prospective study assessing the long-term impact of a short-course of ART (SCART) during PHI. Twenty-eight subjects with defined PHI received 3 months of SCART at HIV-1 seroconversion. HIV-specific interferon-γ+ CD4+ T cell responses, CD4 cell counts and plasma viral loads were assessed prospectively. Clinical outcome was defined as the time taken from PHI to a fall in CD4 cell counts 3 years) preservation of virus-specific CD4+ cells occurred in 45% of patients receiving SCART in PHI. There was no correlation between either the presence or magnitude of these responses and clinical outcome.

Using oligonucleotide primers complementary to conserved regions in the mouse erythropoietin (Epo) gene, a portion of the rat Epo gene was amplified by the polymerase chain reaction to produce a probe suitable for assay of rat Epo mRNA by RNAse protection. The assay, which has sufficient sensitivity to measure to Epo mRNA in unstimulated rat kidneys, was used to demonstrate high amplitude in vitro modulation of Epo mRNA levels in response to changes in perfusate flow rate and oxygen tension in isolated kidneys, thus providing clear evidence that all the necessary events linking changes in oxygen delivery to the modulation of Epo mRNA levels can occur intrarenally.

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is a major obstacle for the design of a successful vaccine. Certain viral polymorphisms encode human leukocyte antigen (HLA)-associated immune escape, potentially overcoming limited vaccine protection. Although transmission of immune escape variants has been reported, the overall extent to which this phenomenon occurs in populations and the degree to which it contributes to HIV-1 viral evolution are unknown. Selection on the HIV-1 env gene at transmission favors neutralization-sensitive variants, but it is not known to what degree selection acts on the internal HIV-1 proteins to restrict or enhance the transmission of immune escape variants. Studies have suggested that HLA class I may determine susceptibility to HIV-1 infection, but a definitive role for HLA at transmission remains unproven. Comparing populations of acute seroconverters and chronically infected patients, we found no evidence of selection acting to restrict transmission of HIV-1 variants. We found that statistical associations previously reported in chronic infection between viral polymorphisms and HLA class I alleles are not present in acute infection, suggesting that the majority of viral polymorphisms in these patients are the result of transmission rather than de novo adaptation. Using four episodes of HIV-1 transmission in which the donors and recipients were both sampled very close to the time of infection we found that, despite a transmission bottleneck, genetic variants of HIV-1 infection are transmitted in a frequency-dependent manner. As HIV-1 infections are seeded by unique donor-adapted viral variants, each episode is a highly individual antigenic challenge. Host-specific, idiosyncratic HIV-1 antigenic diversity will seriously tax the efficacy of immunization based on consensus sequences.

BackgroundGenetic diversity of the human immunodeficiency virus type 1 (HIV-1) population within an individual is lost during transmission to a new host. The demography of transmission is an important determinant of evolutionary dynamics, particularly the relative impact of natural selection and genetic drift immediately following HIV-1 infection. Despite this, the magnitude of this population bottleneck is unclear.ResultsWe use coalescent methods to quantify the bottleneck in a single case of homosexual transmission and find that over 99% of theenvandgagdiversity present in the donor is lost. This was consistent with the diversity present at seroconversion in nine other horizontally infected individuals. Furthermore, we estimated viral diversity at birth in 27 infants infected through vertical transmission and found there to be no difference between the two modes of transmission.ConclusionAssuming the bottleneck at transmission is selectively neutral, such a severe reduction in genetic diversity has important implications for adaptation in HIV-1, since beneficial mutations have a reduced chance of transmission.

Antigenic variation inherent in human immunodeficiency virus type 1 (HIV-1) virions that successfully instigate new infections transferred by sex has not been well defined. Yet this is the viral “challenge” which any vaccine-induced immunity must deal with. Closely timed comparisons of the virus circulating in the “donor” and that which initiates new infection are difficult to carry out rigorously, as suitable samples are very hard to get in the face of ethical hurdles. Here we investigate HIV-1 variation in four homosexual couples where we sampled blood from both parties within several weeks of the estimated transmission event. We analyzed variation within highly immunogenic HIV-1 internal proteins encoding epitopes recognized by cytotoxic Tlymphocytes (CTLs). These responses are believed to be crucial as a means of containing viral replication. In the donors we detected virions capable of evading host CTL recognition at several linked epitopes of distinct HLA class I restriction. When a donor transmitted escape variants to a recipient with whom he had HLA class I molecules in common, the recipient's CTL response to those epitopes was prevented, thus impeding adequate viral control. In addition, we show that even when HLA class I alleles are disparate in the transmitting couple, a single polymorphism can abolish CTL recognition of an overlapping epitope of distinct restriction and so confer immune escape properties to the recipient's seroconversion virus. In donors who are themselves controlling an early, acute infection, the precise timing of onward transmission is a crucial determinant of the viral variants available to compose the inoculum.

Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of “negatively associated” or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.

The outcome of human immunodeficiency virus type 1 (HIV-1) infection is related to the set-point plasma virus load (pVL) that emerges after primary HIV-1 infection ( PHI). This set-point pVL generally remains stable but eventually increases with progression to disease. However, the events leading to loss of viremic control are poorly understood. Here, we describe an individual who presented with symptomatic PHI and subsequently progressed rapidly, after an initial period of 1 year during which viral replication was well controlled. Escalation of viral replication in this atypical case was preceded by the emergence of escape variants in many epitopes targeted by dominant CD8(+) T cell responses and a marked decrease in HIV-1-specific CD4(+) and CD8(+) T cell frequencies. There were no changes in viral tropism, replication kinetics, or neutralizing antibody titers. These findings demonstrate the temporal relationship between viral escape from CD8(+) T cell activity, decrease in HIV-1-specific T cell frequencies, and loss of control of viral replication.

The Journal of Infectious Diseases, 189 (7), ISSN:0022-1899, ISSN:1537-6613

BACKGROUND: According to the "autovaccination hypothesis," reexposure to human immunodeficiency virus (HIV) during treatment interruptions may stimulate the HIV-specific immune response and lead to low viremia after withdrawal of highly active antiretroviral treatment (HAART). Many patients who started HAART earlier in their disease course than is currently recommended would like to discontinue, but it is unknown whether it is safe to do so. OBJECTIVES: To determine whether repeated treatment interruptions of HAART (1) stimulated the cytotoxic HIV-specific immune response and whether such stimulation correlated with low viremia off treatment, and (2) were safe with respect to clinical complications, development of viral resistance, and decline in CD4 cell counts. DESIGN: Interventional study with before-after comparison. SETTING: Outpatient clinics of university hospitals in Switzerland and Spain. PATIENTS: A total of 133 patients receiving HAART, with a median CD4 cell count of 740/ microL, and whose viral load had been undetectable for a median of 21 months. INTERVENTIONS: HAART was interrupted for 2 weeks, restarted, and continued for 8 weeks. After 4 such cycles, treatment was indefinitely suspended 40 weeks after study entry. MAIN OUTCOME MEASURES: HIV-specific cytotoxic T-cell responses were evaluated by interferon gamma enzyme-linked immunospot analysis. The proportion of "responders" (viral load

Potent antiretroviral therapy (ART) suppresses HIV-1 viral replication and results in decreased morbidity and mortality. However, prolonged treatment is associated with drug-induced toxicity, emergence of drug-resistant viral strains, and financial constraints. Structured therapeutic interruptions (STIs) have been proposed as a strategy that could boost HIV-specific immunity, through controlled exposure to autologous virus over limited time periods, and subsequently control viral replication in the absence of ART. Here, we analyzed the impact of repeated STIs on virological and immunological parameters in a large prospective STI study. We show that: ( i ) the plateau virus load (VL) reached after STIs correlated with pretreatment VL, the amount of viral recrudescence during the treatment interruptions, and the off-treatment viral rebound rate; ( ii ) the magnitude and the breadth of the HIV-specific CD8 + T lymphocyte response, despite marked interpatient variability, increased overall with STI. However, the quantity and quality of the post-STI response was comparable to the response observed before any therapy; ( iii ) individuals with strong and broad HIV-specific CD8 + T lymphocyte responses at baseline retained these characteristics during and after STI; ( iv ) the increase in HIV-specific CD8 + T lymphocyte frequencies induced by STI was not correlated with decreased viral set point after STI; and ( v ) HIV-specific CD4 + T lymphocyte responses increased with STI, but were subsequently maintained only in patients with low pretreatment and plateau VLs. Overall, these data indicate that STI-induced quantitative boosting of HIV-specific cellular immunity was not associated with substantial change in viral replication and that STI was largely restoring pretherapy CD8 + T cell responses in patients with established infection.

Background: National and international guidelines call for the treatment of primary HIV infection (PHI) with combination antiretroviral therapy, although the ideal timing and duration of this intervention is unknown. Recent immunological studies of antiretroviral therapy on small numbers of patients with PHI have reported preservation of HIV-specific CD4 T-helper responses, ordinarily lost in the absence of intervention. We sought to investigate whether a short course of antiretroviral therapy (SCART) at PHI was sufficient to preserve HIV-specific cellular immunity.Methods: Forty-five subjects with confirmed PHI were offered SCART at diagnosis. HIV specific cellular immune responses and virological parameters were assessed at monthly intervals.Results: Thirty-seven of the subjects chose SCART at PHI, and achieved a plasma viral load < 50 RNA copies/ml by a median of 10 weeks (range, 4-32 weeks). Two of the 45 individuals had evidence of genotypic HIV drug resistance at baseline, and none developed new mutations following therapy. All patients who received SCART at PHI showed preservation of HIV-specific CD4 T-helper responses up to 64 weeks off SCART.Conclusion: SCART at PHI was safe, did not induce the development of drug resistance, and appeared sufficient to preserve HIV-specific CD4 T-helper responses. However, PHI is highly heterogeneous, and a large-scale randomized trial of SCART at PHI IS now needed. (C) 2002 Lippincott Williams & Wilkins.

The immune response to HIV-1 in patients who carry human histocompatibility leukocyte antigen (HLA)-B27 is characterized by an immunodominant response to an epitope in p24 gag (amino acids 263–272, KRWIILGLNK). Substitution of lysine (K) or glycine (G) for arginine (R) at HIV-1 gag residue 264 (R264K and R264G) results in epitopes that bind to HLA-B27 poorly. We have detected a R264K mutation in four patients carrying HLA-B27. In three of these patients the mutation occurred late, coinciding with disease progression. In another it occurred within 1 yr of infection and was associated with a virus of syncytium-inducing phenotype. In each case, R264K was tightly associated with a leucine to methionine change at residue 268. After the loss of the cytotoxic T lymphocyte (CTL) response to this epitope and in the presence of high viral load, reversion to wild-type sequence was observed. In a fifth patient, a R264G mutation was detected when HIV-1 disease progressed. Its occurrence was associated with a glutamic acid to aspartic acid mutation at residue 260. Phylogenetic analyses indicated that these substitutions emerged under natural selection rather than by genetic drift or linkage. Outgrowth of CTL escape viruses required high viral loads and additional, possibly compensatory, mutations in the gag protein.

Cellular immune responses are likely to play a key role in determining the clinical outcome in acute infection with hepatitis C virus (HCV), but the dynamics of such responses and their relationship to viral clearance are poorly understood. In a previous study we have shown highly activated, multispecific cytotoxic T lymphocyte responses arising early and persisting in an individual who subsequently cleared the virus. In this study the HCV-specific CD8+ lymphocytes response has been similarly analyzed, using peptide-HLA class I tetramers, in a further nine individuals with documented acute HCV infection, six of whom failed to clear the virus. Significant populations of virus-specific CD8+ lymphocytes were detected at the peak of acute hepatic illness (maximally 3.5% of CD8+ lymphocytes). Frequencies were commonly lower than those seen previously and were generally not sustained. Early HCV-specific CD8+ lymphocytes showed an activated phenotype in all patients (CD38+ and HLA class II+), but this activation was short-lived. Failure to sustain sufficient numbers of activated virus-specific CD8+ lymphocytes may contribute to persistence of HCV.

During zidovudine therapy, human immunodeficiency virus type 1 (HIV-1) acquires a distinctive set of mutations that diminish the sensitivity of the virus to this drug in vitro. An AIDS patient is described who, while being treated with zidovudine, transmitted HIV-1 bearing a drug resistance mutation to a young woman who had never received zidovudine treatment. DNA sequencing of HIV-1 proviruses confirmed that these 2 persons shared HIV genetic variants, including a mutation at codon 70 in the reverse transcriptase gene associated with reduced in vitro sensitivity to zidovudine. This mutation persisted in the woman > 1 year in the absence of antiretroviral therapy. HIV-1 with genetic markers of zidovudine resistance can be transmitted heterosexually, but it is uncertain whether dissemination of drug-resistant virus will substantially reduce the usefulness of this drug.

Fifty-two patients who had been bitten by Russell's vipers in Myanmar developed acute renal failure (serum creatinine exceeding 1.3 mg/dL). Thirty-four of them (65%) became oliguric, but the other 18 (35%) maintained a urine output of more than 400 mL/24 h. In oliguric patients, gastrointestinal haemorrhages, renal angle tenderness and conjunctival oedema occurred more commonly, and peak serum creatinine, blood urea nitrogen and the fractional excretion of sodium were significantly higher (P < 0.01) than in non-oliguric patients, indicating a greater degree of renal damage. Urinary concentrations of beta 2 microglobulin and retinol binding protein were raised in most of the patients indicating failure of proximal tubular reabsorption of these proteins, while high urinary N-acetyl glucosaminidase concentrations were consistent with renal tubular damage. Plasma concentrations of active renin were very high, suggesting that renal ischaemia, associated with activation of the renin-angiotensin system, was involved in the development of renal dysfunction.

In Thailand 29 patients were proved to have been bitten by arboreal green pit vipers: 24 by Trimeresurus albolabris and 5 by T. macrops. They were studied in order to define the clinical effects of envenoming, to characterize the haemostatic abnormalities and assess the efficacy of Thai Red Cross antivenom. T. macrops caused only local painful swelling, neutrophil leucocytosis and thrombocytopenia. T. albolabris caused more severe envenoming with local blistering and necrosis, shock, spontaneous systemic bleeding, defibrination, thrombocytopenia and leucocytosis. There was no evidence of disseminated intravascular coagulation, but fibrinolytic activity was increased. Platelet function was normal. The product of admission venom antigen concentration and the delay between bite and admission was significantly higher in defibrinated patients than in those without severe coagulopathy. Antivenom (5 ampoules intravenously) restored blood coagulability, but there was persistent venom antigenaemia, associated in some cases with recurrent coagulopathy. The literature on bites by south Asian green pit vipers of the genus Trimeresurus is reviewed; these bites are common medical problems and causes of morbidity. The identification of individual species is difficult, but may be important if antivenom is to be improved and used appropriately.

To determine the frequency and outcome of hypoglycemia during diarrhea in childhood, we screened 2003 consecutive patients less than 15 years of age who were admitted to a diarrhea treatment center in Dhaka, Bangladesh. Hypoglycemia, defined as a blood glucose concentration less than 2.2 mmol per liter, was found in 91 patients (4.5 percent), 39 (42.9 percent) of whom died. We also measured the plasma concentrations of glucoregulatory hormones and gluconeogenetic substrates in 46 of the patients with hypoglycemia who were 2 to 15 years old and in 25 normoglycemic patients matched with them for age and weight. The patients with hypoglycemia had had diarrhea for less time than the normoglycemic patients (median, 12 vs. 72 hours; P less than 0.05), and their last feeding had been 18 hours before admission, as compared with 9 hours for the normoglycemic patients (P less than 0.05). The groups were similar in terms of nutritional status, the proportion of patients who had fever, and the types of pathogens recovered from stool samples. The plasma C-peptide concentrations were low (less than 0.30 nmol per liter) in all the hypoglycemic patients. As compared with the normoglycemic patients, the patients with hypoglycemia had elevated median plasma concentrations of glucagon (44 vs. 11 pmol per liter; P = 0.001), epinephrine (3400 vs. 1500 pmol per liter; P = 0.012), norepinephrine (7500 vs. 2900 pmol per liter; P = 0.002), and lactate (3.5 vs. 2.1 mmol per liter; P = 0.020) and similar alanine and beta-hydroxybutyrate concentrations. Eighteen hypoglycemic patients with severe malnutrition had been ill longer than 26 better-nourished patients with hypoglycemia (median duration of illness, 18 vs. 10 hours; P = 0.023) and had lower median plasma concentrations of lactate (1.9 vs. 3.9 mmol per liter; P = 0.021) and alanine (173 vs. 293 micromol per liter; P = 0.040). We conclude that hypoglycemia is a major cause of death in association with diarrhea. Because the glucose counterregulatory hormones were appropriately elevated in the children with diarrhea and hypoglycemia, whereas the gluconeogenetic substrates were inappropriately low, we further conclude that the hypoglycemia observed in such patients is most often due to the failure of gluconeogenesis.

Background. CD4 + T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4 + T cell helper responses appear to be absent or short-lived, using functional assays. Methodology/Principal Findings. Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4 + T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4 + T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4 + T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer + CD4 + T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer + cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4 + T cell epitope in patients with evolving chronic hepatitis C. Conclusions/Significance. During acute hepatitis C a CD4 + T cell response against this epitope is readily induced in most, if not all, HLA-DR1 + patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.